Tetrahydropyrazolopyrimidine compounds

ABSTRACT

Embodiments of the disclosure relate to tetrahydropyrazolopyrimidine compounds that act as antagonists or inhibitors for Toll-like receptors 7 and/or 8, and their use in pharmaceutical compositions effective for treatment of systemic lupus erythematosus (SLE) and lupus nephritis.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional PatentApplication No. 61/654,023, filed on May 31, 2012. That application isincorporated by reference herein.

BACKGROUND

1. Field of the Disclosure

Embodiments of the disclosure relate to tetrahydropyrazolopyrimidine(“THPP”) compounds and pharmaceutical agents comprising one or more ofthose compounds as active ingredient(s). More particularly, embodimentsof the disclosure relate to a THPP compound that acts as an antagonistor inhibitor for Toll-like receptors (TLR) 7 and 8, and its use in apharmaceutical composition effective for treatment of systemic lupuserythematosus (SLE) and lupus nephritis.

2. Description of Related Art

Systemic lupus erythematosus (SLE) and lupus nephritis are autoimmunediseases characterized by inflammation and tissue damage. For example,SLE may cause damage to the skin, liver, kidneys, joints, lungs, andcentral nervous system. SLE sufferers may experience general symptomssuch as extreme fatigue, painful and swollen joints, unexplained fever,skin rash, and kidney dysfunction. Because organ involvement differsamongst patients, symptoms may vary. SLE is predominantly a disease ofyounger women, with peak onset between 15-40 years of age and anapproximate 10-fold higher prevalence in women vs. men.

Current treatments for SLE typically involve immunomodulatory drugs suchas hydroxychloroquine, prednisone, and cyclophosphamide. All of thesedrugs may have dose-limiting side effects.

BRIEF SUMMARY OF THE DISCLOSURE

Embodiments of the disclosure provide compounds and methods of use forpreventing or treating diseases or conditions characterized by Toll-likereceptor 7 or 8 activation in patients. One embodiment features acompound of formula (I):

or a pharmaceutically acceptable salt thereof, or a stereoisomer thereofor mixture of stereoisomers thereof, wherein

-   R₁ is optionally substituted piperidinyl, optionally substituted    pyridyl, optionally substituted pyrrolyl, optionally substituted    pyrroldinyl, optionally substituted thiazolyl,    1,4-dimethylthiazolyl, 2-ethyl-4-methylthiazolyl,    2-isopropylthiazol-5-yl, thiazolyl, 3-ethylthiazol-5-yl,    1-methylsulfonylpiperidin-4-yl, or    -   R₁ is —C(O)Z, where Z is optionally substituted piperazinyl,        optionally substituted pyridyl, optionally substituted pyrrolyl,        (S)-2-(3-ethylpiperazin-1-yl), optionally substituted        pyrrolopyrrolyl, piperidin-3-ylamino, or,    -   R₁ is

where R₁₃ is H, optionally substituted pyrazolyl, optionally substitutedimidazolyl, benzyl, 3-hydroxybutyl,3-(dimethylamino)-2,2-dimethylpropyl, amide, methylamide, ethylamide,optionally substituted pyridyl, methylsulfonyl,(1-methylimidazol-2-yl)methyl, (1,5-dimethylimidazol-4-yl)methyl,(1-methylpyrrol-2-yl)methyl, or where R₁₃ is C(O)W, where W is —N(CH₃)₂,optionally substituted piperidinyl, optionally substituted piperazinyl,optionally substituted pyrazolyl, optionally substituted pyrrolyl, oroptionally substituted morpholinyl, or,

-   -   R₁ is

where R₁₄ is —C(O)CH₃, H, or (1-methylpyrrol-2-yl)methyl, or,

-   -   R₁ is

optionally substituted piperidinyl, optionally substituted piperazinyl,optionally substituted pyrazolyl, optionally substituted pyrrolyl, or

-   -   R₁ is

where A, B, and D may all be carbon, or where two of A, B, and D arecarbon and the other is nitrogen, or where one of A, B, and D is carbonand the remaining two are nitrogen; and when A is nitrogen R₄ is absent,when B is nitrogen R₂ is absent, and when D is nitrogen R₃ is absent;and wherein

-   -   R₂ is H, —CH₃, or F, or, with R₃ and the atoms at positions a        and b, forms an optionally substituted pyridine or a pyrazole;        and wherein    -   R₃ is H, F, Cl, —CN, —CH₃, —OCH₃, —OH, —NH₂, methylsulfonyl,

or, with R₄ and the atoms at b and c, forms an optionally substitutedbenzene, optionally substituted imidazole, optionally substitutedpyrazole, optionally substituted pyrazolidine, optionally substitutedimidazolidine, optionally substituted isothiazole,

or, with R₂ and the atoms at a and b, forms an optionally substitutedpyridine or optionally substituted pyrazole; and wherein

-   -   R₄ is F, —CN, —OCH₃, —OEt, H, Cl, Br, —NH—C(O)—CH—(CH₃)₂,        —N(CH₃)₂, —CH₃, —CH₂OH,

optionally substituted piperazinyl, optionally substituted piperidinyl,optionally substituted pyrazolyl, optionally substituted pyrrolyl,4-hydroxypiperizin-1-yl, optionally substituted piperidinyl not attachedto a phenyl group through a nitrogen, or, with R₃ and the atoms at b andc, forms an optionally substituted pyrazole ring or

or, with R₅ and the atoms at c and d, forms an optionally substitutedpyrazole or an optionally substituted pyrrole, or,

-   -   R₄ is -(q)-C(O)X, where q is a single bond, is —NH—, or is        —CH₂—, and    -   where X is —NR₁₁R₁₂,        -   where R₁₁ and R₁₂ are both H, both —CH₂CH₃, or both —CH₃, or            where one of R₁₁ and R₁₂ is H and the other is            1,1-dimethylethyl, cyclobutyl, cyclopropyl, lower alkyl,            methyl alcohol, ethyl alcohol, propyl alcohol,            cyclobutylmethyl; 2,3-dihydroxypropyl, benzyl, azetidinyl,            optionally substituted piperidinyl, optionally substituted            piperazinyl, optionally substituted pyrazolyl, optionally            substituted pyrrolyl, optionally substituted azetidinyl,            —CH₂—NH—CH₃, alcohol, —OCH₃, or

or

-   -   where X is optionally substituted pyrrolidinyl, optionally        substituted piperidinyl, optionally substituted pyrrolidinyl,        optionally substituted piperazinyl, or optionally substituted        morpholinyl,

and wherein

-   R₅ is H, F, Cl, —CH₃, —OCH₃, pyrrolyl,

or, with R₄ and the atoms at c and d, forms an optionally substitutedbenzene, an optionally substituted pyrazole, an optionally substitutedpiperidinyl, an optionally substituted piperazinyl, or an optionallysubstituted pyrrole, or, with R₆ and the atoms at d and e, forms anoptionally substituted pyridine, or R₅ is C(O)Y, where Y is —NH₂,—N(CH₃)₂, optionally substituted piperazinyl, optionally substitutedpiperidinyl, optionally substituted piperidinyl, optionally substitutedpiperazinyl, optionally substituted pyrazolyl, optionally substitutedpyrrolyl,

and wherein

-   R₆ is H, F, —CH₃, —CF₃, or, with R₅ and the atoms at c and d, forms    an optionally substituted benzene or an optionally substituted    pyrazole; and wherein-   R₇ is H, —CF₃, —CHF₂, —CF₂CH₃, —CH₃, or —C(CH₃)₃; and wherein-   R₈ is

and wherein

-   R₉ is Br, Cl, F, I, or H.

In some embodiments of the disclosure one or more provisos are applied.These provisos typically exclude one or more compounds that mightotherwise be included within a stated genus. When reviewing the provisosbelow, it will be understood that the converse is also true. Forexample, if a proviso states that when R₄ is F: then R₂ is not CH₃ or F,then it is also true that when R₂ is selected to be —CH₃ or F, then R₄is not F. It will also be noted that when a proviso is presented as aseries of statements, the later statements are not related unlessdirectly presented as provisos elsewhere in the document. For example,if a proviso states that when R₄ is F: R₂ is not —CH₃ or F; and R₃ isnot —CH₃, then the implication should not be made from that statementalone that when R₃ is —CH₃, then R₂ is not —CH₃ or F.

One or more of the following provisos may be applied in variousembodiments presented herein:

-   -   when R₄ is F: R₂ is not —CH₃ or F; R₃ is not —CH₃, —CN, F, Cl,        or —OCH₃; R₅ is not —CH₃, F, Cl, or —OCH₃; and R₆ is not —CH₃ or        F;    -   when R₄ is Cl: R₂ is not F; R₃ is not F or —CN; R₅ is not F,        —CN, or —C(O)N(CH₃)₂; R₆ is not —CF₃ or F; D is not nitrogen;        and either R₅ is —C(O)NH₂ or one of R₂, R₃, R₅, and R₆ is —CH₃;    -   when R₄ is —CH₃: R₃ is not F; R₅ is not F; and R₅ and R₆ do not        form a pyrimidine together with the atoms at d and e;    -   when R₄ is —OCH₃: R₂ is not F; R₃ is not Cl or —OCH₃, R₅ is not        Cl or —OCH₃; and R₆ is not F or —CF₃;    -   when R₄ is —CN: R₂ is not F; R₃ is not Cl, F, or —OCH₃, R₅ is        not Cl, F, or —OCH₃; and R₆ is not F;    -   when R₄ is —OCH₂CH₃: R₃ is not Cl or F; R₅ is not Cl or F; and        R₆ is not —CF₃;    -   when R₄ is

R₃ is not H or F; and R₅ is not H or F;

-   -   when R₄ is

at least one of R₂, R₃, R₅, and R₆ is not H;

-   -   when R₄ is

R₃ is not F; and R₅ is not F;

-   -   when R₂ is F: R₃ is not —OCH₃ or F; R₅ is not —CN; and at least        one of R₃, R₄, R₅, and R₆ is not H;    -   when R₂ is Cl: R₃ is not F;    -   when R₂ is —CH₃: R₃ is not Cl; at least one of R₃, R₄, R₅, and        R₆ is not —CH₃; and R₄ and R₅ do not form a pyrazolyl with the        atoms at c and d;    -   when R₃ is —OCH₃: R₂ is not F; and R₆ is not F;    -   when R₃ is F: R₂ is not —OCH₃; and X is not

-   -   when R₃ is Cl: R₅ is not Cl; R₁₁ is not benzyl; and R₁₂ is not        benzyl;    -   when R₅ is Cl, R₆ is not —CH₃; R₁₁ is not benzyl; and R₁₂ is not        benzyl;    -   when R₅ is F or —OCH₃: R₆ is not F;    -   when R₆ is F: at least one of R₂, R₃, R₄, and R₅ is not H;    -   when R₃ and R₅ are H: R₁₁ is not cyclopropyl; and R₁₂ is not        cyclopropyl;    -   when R₉ is Cl, R₁ is not an amide group;    -   when B is nitrogen and A and D are carbon: R₄ may not be —CN or

-   -   when R₇ is —CHF₂ and R₄ is

then R₄ does not have the absolute stereochemistry

andwherein, when R₈ is

then the following provisos are in effect:

-   -   when R₄ is F: at least one of R₂, R₃, R₅, and R₆ is not H; R₃ is        not C(O)N(CH₃)₂; and R₅ is not C(O)N(CH₃)₂;    -   when R₄ is Cl: at least one of R₂, R₃, R₅, and R₆ is not H;    -   when R₃ is F: R₄ is not C(O)NHCH₂CH₂CH₂CH₃, C(O)N(CH₃)₂,        C(O)NHCH₂CH₂CH₃, or C(O)NHC(CH₃)₃;    -   R₄ is not C(O)NHCH₂CH₂CH₂OH, C(O)NHCH(CH₃)₂, —CN, or

-   -   R₁ is not

-   -   R₅ is not

-   -   R₃ is not

-   -   when R₂ is F: R₅ is not —C(O)NH₂;    -   when R₂ is —CH₃, R₄ and R₅ do not form a pyrazole with atoms at        c and d; and    -   when B is nitrogen, R₃ and R₄ do not form an optionally        substituted imidazole with the atoms at b and c; and        wherein, when R₈ is

then following provisos are in effect:

-   -   R₄ is not —CH₃, —C(O)NHCH₂CH₂OH, —NHC(O)CH(CH₃)₂, or

-   -   when R₄ is C(O)NHCH₃, at least one of R₂, R₃, R₅, and R₆ is not        H;    -   when R₄ is —OCH₃: R₃ is not F or —CH₃; and R₅ is not F or —CH₃;    -   when R₄ is

R₃ is not Cl; and R₅ is not Cl;

-   -   when R₄ is —C(O)NHCH(CH₃)₂ or —C(O)N(CH₂CH₃)₂: at least one of        R₃ and R₅ is not H;    -   R₅ is not —C(O)NH₂; and    -   R₆ is not —CF₃.

In a further embodiment the compound of formula (I) has the absolutestereochemistry set forth in formula (II):

with the proviso that, when R₈ is

the following provisos are in effect:

-   -   when R₃ is F, R₄ is not

-   -   when R₅ is F, R₄ is not

-   -   when R₅ is —CH₃, R₃ and R₄, with atoms at b and c, do not form

and

-   -   when R₃ is —CH₃, R₄ and R₅, with the atoms at c and d, do not        form

In a further embodiment the compound of formula (I) has the absolutestereochemistry set forth in formula (III):

with the proviso that when R₈ is

R4 is not

andwith the proviso that when R₈ is

the following provisos are in effect:

-   -   when R₂ is —CH₃: R₃ and R₄ do not form an optionally substituted        pyrazolyl with the atoms at b and c;    -   when R₂ is —CH₃: R₄ and R₅ do not form an optionally substituted        pyrazolyl with the atoms at c and d;    -   when R₂ is F: R₄ is not C(O)NH₂;    -   R₃ and R₄, with the atoms at b and c, do not form

-   -   when R₃ is Cl: R₄ is not —C(O)NHCH₃ or —C(O)NH₂;    -   R₃ is not pyrazolyl;    -   when R₃ is F: R₄ is not

or —C(O)NH₂;

-   -   when R₃ is —CH₃: R₄ and R₅ do not form an optionally substituted        pyrazolyl with the atoms at c and d;    -   R₄ is not —C(O)NHCH₂CH₂CH₂OH;    -   R₄ is not —CN or

-   -   when R₅ is —CH₃, R₃ and R₄ do not form an optionally substituted        pyrazolyl with the atoms at b and c;    -   when R₅ is Cl: R₄ is not —C(O)NH₂;    -   when R₅ is F: R₄ is not C(O)NH₂;    -   R₅ is not pyrazolyl;    -   when R₆ is —CH₃: R₄ and R₅ do not form an optionally substituted        pyrazolyl with the atoms at c and d; and    -   when B is nitrogen, R₄ is not —C(O)NHCH₃.

In one embodiment, R₁ is piperidinyl or pyridyl; R₇ is —CF₃; R₈ is

and R₉ is F, Cl, Br, or I.

In another embodiment, R₁ is —C(O)Z, where Z is piperazinyl,piperidinyl, pyrrolopyrrolyl, or piperidinyl propyl; R₇ is —CF₃; R₈ is

and R₉ is H.

In another embodiment, R₁ is

R₇ is —CF₃, R₈ is

and R₉ is H.

In another embodiment, R₁ is

R₂ is H, —CH₃, or, with R₃, forms

R₃ is H or, with R₂, forms

or, with R₄, forms

R₄ is H, —CH₃, —NHC(O)NH₂, or, with R₃, forms

R₅ is H; R₆ is H; R₇ is —CF₃; R₈ is

and R₉ is H.

In another embodiment R₁ is

R₂ is H, F, or —CH₃, R₃ is H or F; R₄ is -(q)-C(O)X, where q is a bondor —CH₂—, and X is piperazinyl attached through a nitrogen to thecarbonyl group of R₄, pyrrolidinyl attached through a nitrogen to thecarbonyl group of R₄, pyrrolopyrrolyl attached through a nitrogen to thecarbonyl group of R₄, azetidinyl attached through a nitrogen to thecarbonyl group of R₄, or

or X is —NR₁₁R₁₂, where one of R₁₁ and R₁₂ is H and the other isoptionally substituted pyrrolidinyl, optionally substituted piperidinyl,optionally substituted piperazinyl, or optionally substitutedazetidinyl; R₅ is H or C(O)Y, where Y is, —NH(CH₃)₂, optionallysubstituted piperazinyl, optionally substituted piperidinyl,

R₆ is H, R₇ is —CHF₂, R₈ is

and R₉ is H.

In a further embodiment, R₁ is

where A, B, and D may all be carbon, or where two of A, B, and D arecarbon and the other is nitrogen, and when A is nitrogen R₄ is absent,when B is nitrogen R₂ is absent, and when D is nitrogen R₃ is absent; R₂is H; R₃ is H or —CH₃; R₄ is —C(O)X, where X is optionally substitutedpiperazinyl, or X is —NR₁₁R₁₂, where R₁₁ and R₁₂ are H, or where one ofR₁₁ or R₁₂ is H, and the other is piperidinyl, pyrrolidinyl, or —CH₃; R₅is —OCH₃, H, or Cl; R₆ is H, R₇ is —CF₃, R₈ is

and R₉ is H.

In a further embodiment, R₁ is

where A, B, and D are carbon;

-   -   R₂ is H, —CH₃, or F, or, with R₃ and the atoms at positions a        and b, forms an optionally substituted pyrazole;    -   R₃ is

or, with R₄ and the atoms at b and c, forms optionally substitutedpyrazole or

or, with R₂ and the atoms at a and b, forms an optionally substitutedpyrazole;

-   -   R₄ is

optionally substituted piperazinyl, or, with R₃ and the atoms at b andc, forms an optionally substituted pyrazole ring or

or, with R₅ and the atoms at c and d, forms an optionally substitutedpyrazole ring, or

-   -   R₄ is -(q)-C(O)X, where q is a bond, and    -   where X is —NR₁₁R₁₂,        -   where R₁₁ and R₁₂ are both H, or where one of R₁₁ and R₁₂ is            H and the other is 1,1-dimethylethyl, cyclobutyl,            cyclopropyl, lower alkyl, C₁₋₃ alcohol, cyclobutylmethyl;            2,3-dihydroxypropyl, benzyl, azetidinyl, pyrrolidinyl,            piperidinyl, methylazetidinyl, pyrazolyl, piperazinyl,            alcohol, —OCH₃, or

or

where X is an optionally substituted piperidinyl attached through anitrogen to the carbonyl group of R₄, optionally substituted piperazinylattached through a nitrogen to the carbonyl group of R₄, optionallysubstituted pyrrolidinyl attached through a nitrogen to the carbonylgroup of R₄, or optionally substituted azetidinyl attached through anitrogen to the carbonyl group of R₄,

and

-   -   R₅ is H, or, with R₄ and the atoms at c and d, forms an        optionally substituted benzene, an optionally substituted        pyrazole, or, with R₆ and the atoms at d and e, forms an        optionally substituted pyridine, or R₅ is C(O)Y, where Y is        —NH₂, —NH(CH₃)₂, optionally substituted piperazinyl attached        through a nitrogen to the carbonyl group of R₅, optionally        substituted piperidinyl attached through a nitrogen to the        carbonyl group of R₅,

and

-   -   R₆ is H, F, —CH₃, or, with R₅ and the atoms at c and d, forms an        optionally substituted pyrazole;    -   R₇ is —CF₃;    -   R₈ is

and R₉ is H.

Another embodiment includes a compound of formula (IV)

-   -   or a pharmaceutically acceptable salt thereof, or a stereoisomer        thereof or mixture of stereoisomers thereof, wherein: R_(7a) is        H or F; and wherein        Ring A is:

wherein Y₁ and Y₂ are independently selected from the group consistingof —CH₂— and —CH₂CH₂—, and wherein each of Y₁ and Y₂ is optionallysubstituted by C₁₋₃ alkyl;

wherein X₁, X₂, and X₃ are independently selected from the groupconsisting of —CH— and N;

wherein X₁, X₂, and X₃ are independently selected from the groupconsisting of —CH— and N;

wherein X is N or —CH— optionally substituted by —CH₃, F, or Cl, and

-   -   wherein R₉ is —C(O)Z, wherein Z is 2,3-dihydroxypropylamine; a        five to seven member cyclic diamine that is optionally bridged        or optionally substituted at a carbon atom with a lower alkyl; a        seven to ten member bicyclodiamine; a seven to eleven member        spirodiamine; —NH substituted with a four to seven member cyclic        amine optionally substituted with —NH₂; —OH; —CH₂NHR, wherein R        is H or lower alkyl; —NH substituted with a seven to eleven        member spiroalkane optionally substituted with —NH₂; or    -   R₉ is CH₃NHC(O)—, and a carbon atom on the aryl ring to which R₉        is attached is substituted with one of —CH₃, F, or Cl; R₉ is        (CH₃)₂CHNHC(O)—, and a carbon atom on the aryl ring to which R₉        is attached is substituted with one of —CH₃, F, or Cl; or R₉ is        (CH₃)₃CNHC(O)— and a carbon atom on the aryl ring to which R₉ is        attached is substituted with one of —CH₃, F, or Cl; or    -   R₉ is

wherein the piperazine is optionally bridged or substituted with loweralkyl and R₁₀ is H or —CH₃; or

-   -   R₉ is

wherein n is 1-3 and the cyclic diamine is optionally bridged orsubstituted with lower alkyl; or

-   -   R₉ is

wherein n is 1-4; or

-   -   R₉ is —NHC(O)NH₂, —CH₂C(O)NH— wherein the nitrogen is        substituted with a four to seven member cyclic amine; —CH₂—C(O)—        wherein the carbonyl is substituted with a seven to ten member        bicyclodiamine; and a four to seven member cyclic amine        substituted with —CH₂C(O)NH₂; or

wherein X is N or —CH— wherein the C is optionally substituted by —CH₃,F, or Cl, and wherein

-   -   R₁₀ is —C(O)NH— wherein the nitrogen is substituted by a four to        seven member cyclic amine; —C(O)— substituted by a seven to ten        member bicyclodiamine; —C(O)— substituted by a seven to eleven        member spirodiamine; pyrazole; [1,2,4]oxadiazole optionally        substituted by —CH₃ on a carbon atom of the oxadiazole;        —NHC(O)CH₃; —CH₂— substituted by a piperazine; —CH₂— substituted        by a piperazine including a methyl substituent; —C(O)—        substituted by a five to seven member cyclic diamine;        —C(O)NHCH₂— wherein the —CH₂— is substituted by azetidine; or        —C(O)— substituted with a five to seven member cyclic amine        wherein the amine includes an —NH₂ substituent; or    -   cyanophenyl; isoquinoline; cyclohexene substituted with —NH₂ at        the 4′ position; 1,4-dimethylindazole-5-yl;        1,6-dimethylindazole-5-yl; cyclohexene substituted with        spiropiperidine at the 4′ position; 1-piperidinopyrazole; or        o-methoxypyridine.

In a further embodiment, a compound or pharmaceutically effective saltof the preceding paragraph of this disclosure has an IC50 less than orequal to 100 nM against human TLR7 receptors expressed in a HEK-293 cellline. In a further embodiment C50 against human TLR7 receptors expressedin a HEK-293 cell line is measured by (1) plating cells of the HEK-293cell line stably expressing TLR7 in Dulbecco's modified Eagle's mediumcontaining 10% fetal bovine serum at a density of 2.22×105 cells/ml intoa 384-well plate and incubating for 2 days at 37° C., 5% CO2; (2) addingthe compound or pharmaceutically acceptable salt thereof and incubatingthe cells for 30 minutes; (3) adding CLO97 (InvivoGen) at 3 ug/ml andincubating the cells for approximately 20 hours; and (4) quantifyingNF-kappaB dependent reporter activation by measuring luminescence.

A further embodiment includes a compound of formula IV, orpharmaceutically effective salt thereof, wherein Ring A is:

wherein X₁, X₂, and X₃ are independently selected from the groupconsisting of —CH— and N;

wherein X₄ is —CH— or N; and wherein Z is piperazine, optionally bridgedor substituted on a carbon by —CH₃; hexahydropyrrolo[3,4]pyrrole; a fourto seven member cyclic amine substituted with —OH or —NH₂; or —NH—substituted with a four to seven member cyclic amine;

wherein X₅ is —CH— or N; and wherein R is pyrazole; [1,2,4]oxadiazoleoptionally substituted by a —CH₃ on a carbon of the oxadiazole; or—C(O)NH— substituted on its nitrogen by a four to seven member cyclicamine;

1,4-dimethylindazole-5-yl; 1,6-dimethylindazole-5-yl;1-piperidinopyrazole; cyclohexene substituted with —NH₂ at the 4′position; cyclohexene substituted with spiropiperidine at the 4′position; or 2-methoxypyridine-4-yl.

In a further embodiment the compound or pharmaceutically effective saltthereof of the preceding paragraph of this disclosure has an IC50 lessthan or equal to 20 nM against human TLR7 receptors expressed in aHEK-293 cell line. In a further embodiment the compound orpharmaceutically effective salt thereof of the preceding paragraph ofthis disclosure has an IC50 less than or equal to 100 nM against humanTLR7 receptors expressed in a HEK-293 cell line. In a further embodimentthe IC50 against human TLR7 receptors expressed in a HEK-293 cell lineis measured by (1) plating cells of the HEK-293 cell line stablyexpressing TLR7 in Dulbecco's modified Eagle's medium containing 10%fetal bovine serum at a density of 2.22×105 cells/ml into a 384-wellplate and incubating for 2 days at 37° C., 5% CO2; (2) adding thecompound or pharmaceutically acceptable salt thereof and incubating thecells for 30 minutes; (3) adding CLO97 (InvivoGen) at 3 ug/ml andincubating the cells for approximately 20 hours; and (4) quantifyingNF-kappaB dependent reporter activation by measuring luminescence.

A further embodiment includes a compound having the absolutestereochemistry set forth in formula (V):

or pharmaceutically acceptable salt thereof, wherein: R_(7a) is H or F;wherein Ring A is:

wherein X₁, X₂, and X₃ are independently selected from the groupconsisting of —CH— and N;

wherein X₄ is —CH— or N; and wherein Z is piperazine, optionally bridgedor substituted on a carbon by —CH₃; hexahydropyrrolo[3,4]pyrrole; a fourto seven member cyclic amine substituted with —OH or —NH₂; or —NH—substituted with a four to seven member cyclic amine;

wherein X₅ is —CH— or N; and wherein R is pyrazole; [1,2,4]oxadiazoleoptionally substituted by a —CH₃ on a carbon of the oxadiazole; or—C(O)NH— substituted on its nitrogen by a four to seven member cyclicamine;

1,4-dimethylindazole-5-yl; 1,6-dimethylindazole-5-yl;

1-piperidinopyrazole; cyclohexene substituted with —NH₂ at the 4′position; cyclohexene substituted with spiropiperidine at the 4′position; or 2-methoxypyridine-4-yl.

In a further embodiment the compound or pharmaceutically effective saltthereof of the preceding paragraph has an IC50 less than or equal to 20nM against human TLR7 receptors expressed in a HEK-293 cell line. In afurther embodiment the compound or pharmaceutically effective saltthereof of the preceding paragraph of this disclosure has an IC50 lessthan or equal to 100 nM against human TLR7 receptors expressed in aHEK-293 cell line. In a further embodiment the IC50 against human TLR7receptors expressed in a HEK-293 cell line is measured by (1) platingcells of the HEK-293 cell line stably expressing TLR7 in Dulbecco'smodified Eagle's medium containing 10% fetal bovine serum at a densityof 2.22×105 cells/ml into a 384-well plate and incubating for 2 days at37° C., 5% CO2; (2) adding the compound or pharmaceutically acceptablesalt thereof and incubating the cells for 30 minutes; (3) adding CLO97(InvivoGen) at 3 ug/ml and incubating the cells for approximately 20hours; and (4) quantifying NF-kappaB dependent reporter activation bymeasuring luminescence.

In further embodiments of the disclosure, compounds have an IC50 againsthuman TLR7 receptors expressed in a HEK-293 cell line less than or equalto 200 nM, less than or equal to 180 nM, less than or equal to 160 nM,less than or equal to 140 nM, less than or equal to 120 nM, less than orequal to 100 nM, less than or equal to 80 nM, less than or equal to 60nM, less than or equal to 40 nM, or less than or equal to 20 nM. Infurther embodiments of the disclosure, compounds have an IC50 againsthuman TLR7 receptors expressed in a HEK-293 cell line from 10 nM to 30nM, from 10 nM to 50 nM, from 10 nM to 100 nM, from 30 nM to 50 nM, from30 nM to 100 nM, or from 50 nM to 100 nM. In further embodiments theIC50 against human TLR7 receptors expressed in a HEK-293 cell line ismeasured by (1) plating cells of the HEK-293 cell line stably expressingTLR7 in Dulbecco's modified Eagle's medium containing 10% fetal bovineserum at a density of 2.22×105 cells/ml into a 384-well plate andincubating for 2 days at 37° C., 5% CO2; (2) adding the compound orpharmaceutically acceptable salt thereof and incubating the cells for 30minutes; (3) adding CLO97 (InvivoGen) at 3 ug/ml and incubating thecells for approximately 20 hours; and (4) quantifying NF-kappaBdependent reporter activation by measuring luminescence.

Further embodiments provide methods for treatment of a systematic lupuserythematosus or lupus including administering a pharmaceuticallyeffective amount of a compound or pharmaceutically acceptable salt ofthe disclosure.

Further embodiments provide methods for antagonizing TLR7, includingadministering a pharmaceutically effective amount of a compound orpharmaceutically acceptable salt of the disclosure.

Further embodiments provide methods for antagonizing TLR8, includingadministering a pharmaceutically effective amount of a compound orpharmaceutically acceptable salt of the disclosure.

Further embodiments provide pharmaceutical compositions comprising atleast one compound or pharmaceutically acceptable salt of the disclosureand at least one pharmaceutically acceptable carrier.

Further embodiments provide methods for treatment of a systematic lupuserythematosus or lupus, including administering a pharmaceuticallyeffective amount of a compound or pharmaceutically acceptable salt ofthe disclosure.

Further embodiments provide methods for antagonizing TLR7, includingadministering a pharmaceutically effective amount of a compound orpharmaceutically acceptable salt of the disclosure.

Further embodiments provide methods for antagonizing TLR8, includingadministering a pharmaceutically effective amount of a compound orpharmaceutically acceptable salt of the disclosure.

Further embodiments provide pharmaceutical compositions comprising atleast one compound or pharmaceutically acceptable salt of the disclosureand at least one pharmaceutically acceptable carrier.

The term “optionally substituted,” as used herein, means that thesubject structure may include, but is not required to include, one ormore substituents independently selected from lower alkyl, methoxy-,—OH, —NH₂, —CH₂—NH—CH₂, —OCH₂CH₂CH₃, or —OCH(CH₃)₂. If the optionallysubstituted moiety is cyclic, then the optional substitution may be amethyl bridge between two atoms in the ring.

The symbol “C(O)” as used herein refers to a carbonyl group having theformula C═O.

Unless otherwise specified, “a” and “an” as used in this disclosure,including the claims, mean “one or more.”

As used herein, “lower alkyl” refers to straight, or, in the case ofthree- and four-carbon groups, straight, branched, or cyclic saturatedhydrocarbons having between one and four carbon atoms.

As used herein, the term “attached through a nitrogen” when referring toa heterocyclic moiety including nitrogen, means that a point ofattachment of the moiety to another structure is through a nitrogen thatis part of the heterocycle.

As used herein, the term “TLR7/8” means “TLR7 and TLR8” or “TLR7 orTLR8” or “TLR7 and/or TLR8.” The particular meaning can be understood bya person skilled in the art based upon the context in which “TLR7/8”appears.

Heterocyclic moieties recited herein include azetidinyl, pyrrolidinyl,piperidinyl, methylazetidinyl, pyrazolyl, piperazinyl, morpholinyl,thiazolyl, pyrrolopyrrolyl, imidazolidinyl, and isothiazolyl. Where aheterocyclic group is mentioned, unless otherwise indicated it will beunderstood that the heterocyclic atom(s) in the group may be at anyposition in the group. It will further be understood that imidazolyl,pyrazolyl, thiazolyl, and pyrrolyl may be unsaturated or partiallyunsaturated. An embodiment of the disclosure may include apharmaceutical composition that includes one or more compounds of thedisclosure with a pharmaceutically acceptable excipient. Thesepharmaceutical compositions may be used to treat or prevent a disease orcondition characterized by TLR7/8 activation in a patient, typically ahuman patient, who has or is predisposed to have such a condition ordisease. Examples of diseases or conditions characterized by TLR7/8activation include systemic lupus erythematosus (SLE) and lupusnephritis.

As used herein, “effective amount” of a compound of an embodiment of thedisclosure is effective amount of the above-identified compounds in anamount sufficient to treat or prevent SLE and lupus nephritis.

Embodiments presented herein may include asymmetric or chiral centers.Embodiments include the various stereoisomers and mixtures thereof.Individual stereoisomers of compounds of embodiments of the disclosuremay be prepared synthetically from commercially available startingmaterials that contain asymmetric or chiral centers, or by preparationof mixtures of enantiomeric compounds followed by resolution of thosecompounds. Suitable methods of resolution include attachment of aracemic mixture of enantiomers, designated (+/−), to a chiral auxiliary,separation of the resulting diastereomer by chromatography orrecrystallization and separation of the optically pure product from theauxiliary; or direct separation of the mixture of optical enantiomers onchiral chromatographic columns.

Embodiments of the disclosure also include a pharmaceutical compositionincluding any compound of the disclosure as well as a pharmaceuticallyacceptable excipient. The pharmaceutical compositions can be used totreat or prevent SLE and lupus nephritis. Therefore, embodiments of thedisclosure may also feature a method for treating or preventing SLE orlupus nephritis in a human patient having or predisposed to having lupusnephritis or SLE.

Embodiments of the disclosure include pharmaceutically acceptable saltsof the compounds presented herein. The term “pharmaceutically acceptablesalt” refers to those salts that are, within the scope of sound medicaljudgment, suitable for use in contact with the tissues of humans andanimals without undue toxicity, irritation, or allergic response.Pharmaceutically acceptable salts are well known in the art. Forexample, S. M. Berge, et al., describes pharmaceutically acceptablesalts in detail in J. Pharmaceutical Sciences 66:1-19, 1977. Salts canbe prepared in situ during final isolation and purification of acompound or separately by reacting a free base group with a suitableorganic acid. Representative acid addition salts include acetate,adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate,bisulfate, borate, butyrate, camphorate, camphersulfonate, citrate,cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate,fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptonate,hexanoate, hydrobromide, hydrochloride, hydroiodide,2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, laurylsulfate, malate, maleate, monomaleate, malonate, methanesulfonate,2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate,pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate,pivalate, propionate, stearate, succinate, sulfate, tartrate,thiocyanate, toluenesulfonate, trifluoroacetate, undecanoate, valeratesalts, and the like. Representative alkali or alkaline earth metal saltsinclude sodium, lithium, potassium, calcium, magnesium and the like, aswell as nontoxic ammonium, quaternary ammonium, and amine cations,including, but not limited to ammonium, tetramethylammonium,tetraethylammonium, methylamine, dimethylamine, trimethylamine,triethylamine, ethylamine, and the like. The term “pharmaceuticallyacceptable ester,” as used herein, represents esters that hydrolyze invivo and include those that break down readily in the human body toleave the parent compound or a salt thereof. Suitable ester groupsinclude, for example, those derived from pharmaceutically acceptablealiphatic carboxylic acids, particularly alkenoic, alkenoic,cycloalkanoic, and alkanedioic acids, in which each alkyl or alkenylgroup typically has not more than 6 carbon atoms. Examples of particularesters include formates, acetates, propionates, butyates, acrylates, andethylsuccinates.

In this application enantiomers are designated by the symbols “R” or “S”or are drawn by conventional means with a bolded line defining asubstituent above the plane of the page in three-dimensional space and ahashed or dashed line defining a substituent beneath the plane of theprinted page in three-dimensional space. If no stereochemicaldesignation is made, then the structure definition includes bothstereochemical options.

BRIEF SUMMARY OF THE FIGURES

FIG. 1 shows short-term in vivo potency against the TLR7 pathway inmouse for compound ER-892887 (which has the chemical name(4-((5S,7R)-5-(3,4-dimethoxyphenyl)-7-(trifluoromethyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidin-2-yl)phenyl)(piperazin-1-yl)methanone).Figure Legend: Female BALB/c mice were dosed by oral gavage with Vehiclealone (0.5% aqueous methyl-cellulose) or compound ER-892887 formulatedin Vehicle at 100 mg/kg or 300 mg/kg. At 13 or 24 hours following oraldosing, mice were injected subcutaneously with 15 ug R848 to stimulateTLR7. Blood plasma was collected by cardiac puncture, and the IL-6 levelat 1.5 hours after TLR7 stimulation was then assessed by standard ELISAprocedure. Percent suppression indicated is relative to IL-6 inductionfollowing Vehicle control dosing. Statistical significance wasdetermined by Mann-Whitney test.

FIG. 2A through FIG. 2D shows results of testing compound ER-892887 inthe BXSB-Yaa strain lupus disease model. Figure Legend: Twelve week oldBXSB-Yaa mice were randomized into groups with equivalent mediananti-dsDNA titers and treated once-a-day orally with Vehicle (Veh; 0.5%methyl-cellulose) alone or 300 mg/kg ER-892887 for 14 weeks total. (FIG.2A) All mice were sacrificed at 26 weeks of age and final anti-dsDNA,anti-Sm/nRNP, and anti-RiboP titers were evaluated by ELISA as comparedto titers observed in 6 wk old pre-diseased mice. (FIG. 2B)Approximately one week prior to sacrifice (25 weeks of age, 13 weeks oftreatment), mice were housed 1-2 per cage in metabolic cages for 18hours to collect urine, and the Urinary Albumin Creatinine Ratio (UACR,proteinuria) was determined for each animal as an indirect measure ofkidney function. (FIG. 2C) At the time of sacrifice, kidneys werecollected from individual mice, fixed in 10% formalin for 24 hours,embedded in paraffin, and H&E stained sections were generated forhistopathology assessment in a blinded fashion. (FIG. 2D) Summary ofmortality observed in the SLE-8 study. Statistical significance wasdetermined by Mann-Whitney test for (A).

FIG. 3A through FIG. 3E show results of testing compound ER-892887 inthe NZBxNZW strain lupus disease model. Figure Legend: Female NZBWF1/Jmice were received at 6 weeks of age, baseline bleeds were performed,and mice were monitored for disease progression by following anti-dsDNAtiters. At 20 weeks of age, mice were randomized into groups withequivalent median anti-dsDNA titers and treated at 23 weeks of age withVehicle (Veh; 0.5% methyl-cellulose) alone or 33, 100, or 300 mg/kgER-892887 once-a-day orally (QD PO). All mice were sacrificed at 45weeks of age (22 weeks total treatment) and blood plasma anti-dsDNAtiters were determined by ELISA. (FIG. 3A) Impact of ER-892887 treatmentover time on anti-dsDNA titers. (FIG. 3B) Bar graph of +22 weekstreatment data for ER-892887 derived from data shown in (A). (FIG. 3C)Just prior to termination at 45 weeks of age (following 22 weeks oftreatment), urine was collected from individual mice, and the UrinaryAlbumin Creatinine Ratio (UACR, proteinuria) was determined for eachanimal as an indirect measure of kidney function. (FIG. 3D) Summary ofmortality observed in this study. (FIG. 3E) At the time of sacrifice,kidneys were collected from individual mice, fixed in 10% formalin for24 hours, embedded in paraffin, and H&E stained sections were generatedfor histopathology assessment in a blinded fashion. Statisticalsignificance was determined by Mann-Whitney test for (C).

FIG. 4A through FIG. 4F show results of additional testing of compoundER-892887 in the NZBxNZW strain lupus disease model. Figure Legend.Female NZBWF1/J mice were received at 8 weeks of age, baseline bleedswere performed, and mice were monitored for disease progression byfollowing anti-dsDNA titers. At 24 weeks of age, mice were randomizedinto groups with equivalent median anti-dsDNA titers and treated at 25weeks of age QD PO with Vehicle (Veh; 0.5% methyl-cellulose) alone or33, 100, or 300 mg/kg ER-892887. Anti-dsDNA titers were determined byELISA on blood plasma samples at the time points indicated. (FIG. 4A)Impact of compound treatment on median anti-dsDNA titers over time up to+17 weeks of treatment. (FIG. 4B) Impact of compound treatment on mediananti-dsDNA at the +13 weeks of treatment time point only from data shownin (A). (FIG. 4C) After 13 weeks (top graph) or 17 weeks (bottom graph)of compound treatment, urine was collected from individual mice, and theUrinary Albumin Creatinine Ratio (UACR, proteinuria) was determined foreach animal as an indirect measure of kidney function. Mice indicatedwithin the circle with high UACR were those animals that died betweenthe +13 wks and +17 wks of treatment (statistical significance wasdetermined by Mann-Whitney test). (FIG. 4D) Blood urea nitrogen (BUN)measured in the plasma from select groups after 13 weeks of treatment.(FIG. 4E) Mortality curves for Vehicle versus compound treated mice upto 17 weeks of treatment. Mortality curve analysis out to +17 wkstreatment indicated that 33 mg/kg and 100 mg/kg ER-892887 treatmentafforded statistically significant survival benefit vs. Vehicle asdetermined by Log-rank (Mantel-Cox) test. (FIG. 4F) Summary of mortalityobserved in this study.

FIG. 5A through FIG. 5D show results of testing compound ER-892887 inthe Pristane: DBA/1 strain lupus disease model. Figure Legend: FemaleDBA/1 mice at 10-11 weeks of age were given an intraperitoneal injectionof 0.5 ml pristane or PBS. Once-a-day oral dosing with Vehicle (Veh;0.5% methyl-cellulose) or 300 mg/kg ER-892887 was begun 3.5 months afterpristane injection for a total of 3 months treatment. (FIG. 5A) Micewere euthanized after 3 months of compound treatment, and anti-dsDNA andanti-RiboP titers were measured in blood plasma samples by ELISA(statistical significance determined by Mann-Whitney test). (FIG. 5B)Arthritis development was assessed monthly by visual scoring. (FIG. 5C)The expression of IFN-regulated genes in whole blood was measured by aqPCR panel after 3 months of treatment, and an IFN gene signature scorewas calculated (see Pharmacology Materials and Methods section fordetails regarding IFN score calculation). (FIG. 5D) Full list of 21genes significantly upregulated by pristane treatment vs. PBS controlsand individual genes which were significantly reduced by ER-892887(Student's t-test).

FIG. 6A through FIG. 6G show results of additional testing of compoundER-892887 in the Pristane: DBA/1 strain lupus disease model. FigureLegend: Female DBA/1 mice at 11-12 weeks of age were given anintraperitoneal injection of 0.5 ml of pristane or PBS. Once-a-day oraldosing with Vehicle (Veh; 0.5% methyl-cellulose) or 33, 100, or 300mg/kg ER-892887 was begun 2 months after pristane injection for a totalof 3 months treatment. Mice were euthanized after 3 months of compoundtreatment, and anti-dsDNA (FIG. 6A), —RiboP (FIG. 6B), —Sm/nRNP (FIG.6C), and -Histone (FIG. 6D) titers were measured in blood plasma samplesby ELISA (statistical significance determined by Mann-Whitney test).(FIG. 6E) Summary of mortality observed in the study during 3 months ofcompound treatment. (FIG. 6F) The expression of IFN-regulated genes inwhole blood was measured by a qPCR panel at the last time point forVehicle and 300 mg/kg ER-892887 treated mice, and an IFN gene signaturescore was calculated (see Pharmacology Materials and Methods section fordetails regarding IFN score calculation). (FIG. 6G) Full list of 22genes significantly upregulated by pristane treatment vs. PBS controlsand individual genes which were significantly reduced by ER-892887(Student's t-test).

FIG. 7A through FIG. 7D show results of testing compound ER-885454 inthe BXSB-Yaa strain lupus disease model. Figure Legend: Nine week oldmale BXSB-Yaa mice were treated once-a-day orally with Vehicle (Veh;0.5% methyl-cellulose) alone or 100 mg/kg or 300 mg/kg ER-885454 for 15weeks total. All mice were sacrificed at 24 weeks of age and finalanti-dsDNA (FIG. 7A) and anti-Sm/RNP (FIG. 7B) titers were evaluated byELISA. (FIG. 7C) Approximately one week prior to sacrifice, mice werehoused 1-2 per cage in metabolic cages for 18 hours to collect urine,and the Urinary Albumin Creatinine Ratio (UACR, proteinuria) wasdetermined for each animal as an indirect measure of kidney function.(FIG. 7D) At the time of sacrifice, kidneys were collected fromindividual mice, fixed in 10% formalin for 24 hours, embedded inparaffin, and H&E stained sections were generated for histopathologyassessment in a blinded fashion. Statistical significance was determinedby Mann-Whitney test for (A), (B), and (C) above.

FIG. 8A through FIG. 8D show results of additional testing of compoundER-885454 in the BXSB-Yaa strain lupus disease model. Figure Legend:Seventeen week old BXSB-Yaa mice were randomized into two groups withequivalent median anti-dsDNA titers and treated once-a-day orally withVehicle (Veh; 0.5% methyl-cellulose) alone or 300 mg/kg ER-885454 for 13weeks total. All mice were sacrificed at 30 weeks of age and finalanti-dsDNA (FIG. 8A) and anti-Sm/RNP (FIG. 8B) titers were evaluated byELISA as compared to titers observed in 7 wk old pre-diseased mice.(FIG. 8C) Approximately one week prior to sacrifice, mice were housed1-2 per cage in metabolic cages for 18 hours to collect urine, and theUrinary Albumin Creatinine Ratio (UACR, proteinuria) was determined foreach animal as an indirect measure of kidney function. (FIG. 8D) At thetime of sacrifice, kidneys were collected from individual mice, fixed in10% formalin for 24 hours, embedded in paraffin, and H&E stainedsections were generated for histopathology assessment in a blindedfashion. Statistical significance was determined by Mann-Whitney testfor (A), (B), and (C) above.

FIG. 9A through FIG. 9C show results of testing compound ER-885454 inthe NZBxNZW strain lupus disease model. Figure Legend: Female NZBWF1/Jmice were received at 6 weeks of age, baseline bleeds were performed,and mice were monitored for disease progression by following anti-dsDNAtiters. At 24 weeks of age, mice were randomized into groups withequivalent median anti-dsDNA titers and treated with Vehicle (Veh; 0.5%methyl-cellulose) alone or 300 mg/kg ER-885454 once-a-day orally (QDPO). All mice were sacrificed at 47 weeks of age (23 weeks totaltreatment) and blood plasma anti-dsDNA titers were determined by ELISA(FIG. 9A). (FIG. 9B) Just prior to termination at 47 weeks of age, urinewas collected from individual mice, and Urinary Albumin Creatinine Ratio(UACR, proteinuria) was determined for each animal as an indirectmeasure of kidney function. (FIG. 9C) At the time of sacrifice, kidneyswere collected from individual mice, fixed in 10% formalin for 24 hours,embedded in paraffin, and H&E stained sections were generated forhistopathology assessment in a blinded fashion. Statistical significancewas determined by Mann-Whitney test for (A) above.

FIG. 10 shows an ORTEP representation of the X-ray structure of compoundER-887006.

FIG. 11A through FIG. 11NN show structures and corresponding chemicalnames according to various embodiments presented herein. “ER-Number” isa reference number assigned to each compound. Where available, activityagainst a HEK cell line stably expressing human TLR7, activity against aHEK cell line stably expressing human TLR9, 1H NMR data, and massspectrometry data are also included.

FIG. 12A through FIG. 12F show the effect of ER-892887 and two commonlyused human lupus treatments in the NZBxNZW strain lupus disease model.Figure Legend: Female NZBWF1/J mice were received at 4 weeks of age,baseline bleeds were performed at 10 and 26 weeks, and mice weremonitored for disease progression by following anti-dsDNA titers andproteinuria. At 26 weeks of age, mice were randomized into groups withequivalent median anti-dsDNA titers and treated with Vehicle (Veh; 0.5%methyl-cellulose) alone or 100 mg/kg ER-892887, 100 mg/kghydroxychloroquine, 0.1 mg/kg prednisolone or 0.5 mg/kg prednisolone,alone or in the combinations indicated, once-a-day orally (QD PO). Allmice were sacrificed at 45 weeks of age (19 weeks of drug treatment) andblood plasma anti-dsDNA titers were determined by ELISA. (FIG. 12A)Impact of ER-892887 treatment on anti-dsDNA titers at termination. (FIG.12B) At 41 weeks of age (following 15 weeks of treatment), urine wascollected from individual mice, and the Urinary Albumin Creatinine Ratio(UACR, proteinuria) was determined for each animal as an indirectmeasure of kidney function. (FIG. 12C) Urine was later collected fromindividual mice just prior to termination at 45 weeks of age, following19 weeks of treatment, and the Urinary Albumin Creatinine Ratio (UACR,proteinuria) was determined for each animal. (FIG. 12D, FIG. 12E, FIG.12F) Survival curves observed in this study during treatment with E6887,each standard treatment comparator (FIG. 12D, hydroxychloroquine; FIG.12E, 0.1 mg/kg prednisolone; FIG. 12F, 0.5 mg/kg prednisolone), or thecombination of E6887 and one of the comparators. Treated groups testedversus vehicle by Mantel-Cox.

FIG. 13A through FIG. 13E show results of testing compound ER-892887 andthree commonly used human lupus treatments in the Pristane: DBA/1 strainlupus disease model. Figure Legend: Female DBA/1 mice at 11 weeks of agewere given an intraperitoneal injection of 0.5 ml pristane or PBS.Once-a-day oral dosing with Vehicle (Veh; 0.5% methyl-cellulose) or 300mg/kg ER-892887, 100 mg/kg hydroxychloroquine, 1 mg/kg prednisolone or100 mg/kg mycophenolate was begun 2 months after pristane injection andcontinued for 3 months of treatment. (FIG. 13A) At the end of the 3month treatment arthritis development was assessed by visual scoring ofswelling and inflammation. (FIG. 13B) Mice were euthanized after 3months of compound treatment, and anti-dsDNA, anti-histone, anti-Sm/RNPand anti-RiboP titers were measured in blood plasma samples by ELISA(statistical significance determined by Mann-Whitney test). ELISA valuesfalling above or below the range of the standard curve were assigned avalue equal to the highest or lowest valid measurement, as appropriate.(FIG. 13C, FIG. 13D, and FIG. 13E, with FIG. 13D being a continuation ofFIG. 13C) The expression of IFN-regulated genes in whole blood wasmeasured by a qPCR panel after 3 months of treatment. The full list ofinterferon-regulated genes significantly upregulated by pristanetreatment vs. PBS controls is shown, and individual genes that weresignificantly reduced by ER-892887 (Student's t-test) are marked in boldwith an asterisk.

FIG. 14A, FIG. 14B, and FIG. 14C present the effect of compoundER-892887 on arthritis and autoantibodies in the Pristane: DBA/1 strainlupus disease model. Figure Legend: Female DBA/1 mice at 10 weeks of agewere given an intraperitoneal injection of 0.5 ml pristane or PBS. At 22weeks of age, mice were randomized into groups with equivalent mediananti-titers and treated with Vehicle (Veh; 0.5% methyl-cellulose) aloneor 300 mg/kg ER-892887 once-a-day orally (QD PO), Arthritis symptoms(swelling and inflammation) were recorded monthly by blinded observers.All mice were sacrificed at 34 weeks of age (12 weeks of treatment) andblood plasma autoantibody titers were determined by ELISA. Prior totermination paws were x-rayed and scored for bone damage and erosions bytwo blinded analysts. (FIG. 14A) Top—Arthritis scores were calculatedmonthly in vehicle and compound dosed-groups. (FIG. 14A) Bottom—X-rayscores were calculated after three months of treatment. (FIG. 14B andFIG. 14C) Autoantibody titers were measured in terminal blood plasmasamples by ELISA (statistical significance determined by Mann-Whitneytest).

DETAILED DESCRIPTION OF THE DISCLOSURE

In addition to their role as innate immune receptors capable ofdetecting exogenous (“non-self”) pathogen-associated molecular patterns(PAMPs—i.e., bacterial LPS detection by TLR4), mammalian Toll-likereceptors (TLRs) are also capable of recognizing endogenous stimuli(DAMPs) released following host tissue damage or stress. Kono, H. and K.L. Rock, How dying cells alert the immune system to danger. Nat RevImmunol, 2008, 8(4): p. 279-89. In the last decade an appreciation forthe link between TLR activation by endogenous (“self”) danger-associatedmolecular patterns (DAMPs) and the etiology of autoimmune disorders hasemerged. Specifically, TLR7 can be activated by single-stranded RNA(ssRNA) derived from both mammalian and viral sources, whereas TLR9 canbe activated by DNA derived from mammalian, viral, and bacterialsources.

Lupus is characterized by auto-antibodies reactive againstdouble-stranded DNA (dsDNA) itself and associated proteins (histones) aswell as against a broad array of RNA-associated proteins such as Ro, La,Smith (Sm), and U1 snRNP. Kirou, K. A., et al., Activation of theinterferon-alpha pathway identifies a subgroup of systemic lupuserythematosus patients with distinct serologic features and activedisease. Arthritis Rheum, 2005. 52(5): p. 1491-503. A second commonhallmark of lupus, which was shown to correlate directly with diseaseseverity, is dysregulated expression of type-1 interferons (IFNs), inparticular IFNα, and the corresponding elevation of a large panel ofIFNα-regulated genes in lupus patients' PBMC (the so called “type-1 IFNgene signature”). Kirou, K. A., et al., supra. A major source of IFN inthe blood is a specialized immunocyte called a plasmacytoid dendriticcell (pDC), which constitutively expresses both TLR7 and TLR9.

A causal relationship between these two disease characteristics,auto-antibodies and IFN levels, was postulated when a number of researchgroups collectively demonstrated that antibody complexes isolated fromlupus patients but not from healthy donors are capable of driving IFNproduction by pDC in a TLR7/9- and RNA/DNA-dependent manner. Means, T.K., et al., Human lupus autoantibody-DNA complexes activate DCs throughcooperation of CD32 and TLR9. J Clin Invest, 2005. 115(2): p. 407-17;Vollmer, J., et al., Immune stimulation mediated by autoantigen bindingsites within small nuclear RNAs involves Toll-like receptors 7 and 8. JExp Med, 2005. 202(11): p. 1575-85; Savarese, E., et al., U1 smallnuclear ribonucleoprotein immune complexes induce type I interferon inplasmacytoid dendritic cells through TLR7. Blood, 2006. 107(8): p.3229-34. Moreover, IFN stimulates increased TLR7/9 expression onB-cells, thereby enhancing TLR/BCR (B-cell receptor) activation ofauto-reactive B-cells to differentiate to antibody-producing plasmacells. Banchereau, J. and V. Pascual, Type I interferon in systemiclupus erythematosus and other autoimmune diseases. Immunity, 2006.25(3): p. 383-92; In this fashion, levels of auto-antibody complexescontaining nucleic acid TLR7/9 ligands drive the pro-inflammatory cycleand lupus disease progression. Type-1 IFN alone has been reported toinduce lupus-like symptoms in humans. Ho, V., et al., Severe systemiclupus erythematosus induced by antiviral treatment for hepatitis C. J.Clin Rheumatol, 2008. 14(3):166-8. Ronnblom L. E., et al. Possibleinduction of systemic lupus erythematosus by interferon-alpha treatmentin a patient with a malignant carcinoid tumor. J. Internal Med. 1990.227:207-10. We believe it is likely that pharmacological antagonism ofTLR7/8 will offer therapeutic benefit to lupus patients by disruptingthis pro-inflammatory cycle, decreasing IFN levels, and dampening theautoimmune disease process mediated by pDC and B-cells.

Several other lines of evidence suggest a role for TLR7 in human lupusetiology and support the notion that TLR receptors are valid targets fordisease intervention. Specific polymorphisms in the 3′ UTR of TLR7 havebeen identified and shown to correlate with both elevated TLR7expression and enhanced IFN gene signature. Shen, N., et al.,Sex-specific association of X-linked Toll-like receptor 7 (TLR7) withmale systemic lupus erythematosus. Proc Natl Acad Sci USA, 2010.107(36): p. 15838-43. In addition, lupus standard-of-care (SOC)anti-malarial drugs such as chloroquine disrupt endosomal TLR7/9signaling and inhibit PBMC and/or pDC IFNα production induced byssRNA-ribonucleoprotein complexes or lupus patient serum. Moreover,myeloid DC and monocytes produce IL-12p40, TNFα, and IL-6 followingself-RNA/TLR8 signaling, suggesting the additional contribution ofTLR8-dependent pro-inflammatory cytokines to human lupus etiology inaddition to TLR7-driven IFN by pDC. Vollmer, supra; Gorden, K. B., etal., Synthetic TLR agonists reveal functional differences between humanTLR7 and TLR8. J Immunol, 2005. 174(3): p. 1259-68.

Mouse model evidence also exists for the role of TLR in lupus. Publishedstudies have collectively demonstrated that both single TLR7 or dualTLR7/9 gene deletion or dual TLR7/9 pharmacologic inhibition reducesdisease severity in four distinct lupus models. Nickerson, K. M., etal., TLR9 regulates TLR7- and MyD88-dependent autoantibody productionand disease in a murine model of lupus. J Immunol, 2010. 184(4): p.1840-8; Fairhurst, A. M., et al., Yaa autoimmune phenotypes areconferred by overexpression of TLR7. Eur J Immunol, 2008. 38(7): p.1971-8; Deane, J. A., et al., Control of toll-like receptor 7 expressionis essential to restrict autoimmunity and dendritic cell proliferation.Immunity, 2007. 27(5): p. 801-10; Savarese, E., et al., Requirement ofToll-like receptor 7 for pristane-induced production of autoantibodiesand development of murine lupus nephritis. Arthritis Rheum, 2008. 58(4):p. 1107-15. Highlighting the role of TLR7 as a critical determinant ofautoimmunity, transgenic overexpression of TLR7 alone leads tospontaneous anti-RNA auto-reactivity and nephritis in the normallydisease-resistant C57BL/6 strain. Deane, supra.

From a safety perspective, there are no reports that TLR7, 8, or9-single or 7/8- and 7/9-dual gene deficient mice are immune-compromisedto the extent that infection by opportunistic pathogens is observed.Likewise, SOC anti-malarials are thought to be largely safe andeffective for long-term use in humans to control lupus disease flare atdoses predicted to at least partially inhibit TLR7/9 signaling.Lafyatis, R., M. York, and A. Marshals-Rothstein, Antimalarial agents:closing the gate on Toll-like receptors? Arthritis Rheum, 2006. 54(10):p. 3068-70; Costedoat-Chalumeau, N., et al., Low blood concentration ofhydroxychloroquine is a marker for and predictor of diseaseexacerbations in patients with systemic lupus erythematosus. ArthritisRheum, 2006. 54(10): p. 3284-90. In fact, save for increasedsusceptibility to Gram-positive bacterial infections in childhood and toa lesser extent in adulthood, humans with highly compromised TLR andIL-1R signaling pathways (MyD88- or IRAK-4-deficiency) are nonethelesshealthy and maintain sufficient host defense mechanisms. Casanova, J.L., L. Abel, and L. Quintana-Murci, Human TLRs and IL-1Rs in HostDefense: Natural Insights from Evolutionary, Epidemiological, andClinical Genetics. Annu Rev Immunol, 2010.

Based on this and other information, we believe that TLR7 in particularis a well-validated target in the context of mouse pre-clinical SLEmodels. Both genetic and functional human studies support the hypothesisthat antagonism of the TLR7 and/or TLR8 pathways will afford therapeuticbenefit to lupus patients. Moreover, both mouse TLR gene deletionstudies and the long-term use of anti-malarials in humans suggest thatpharmacological TLR7, 8 and/or 9 suppression can be undertaken withoutsignificantly compromising host defense.

A compound that suppresses TLR7, TLR8, or both TLR7 and TLR8 maytherefore be expected to act as a therapeutic or prophylactic agent forSLE or lupus nephritis.

The present inventors have found compounds that suppress TLR 7 and/or 8and are therefore expected to have a prophylactic or therapeutic effecton SLE or lupus nephritis. Compounds and methods of the disclosure aredescribed herein.

II. Therapeutic Use

Dosage levels of active ingredients in the pharmaceutical compositionsof the disclosure may be varied to obtain an amount of the activecompound(s) that achieves the desired therapeutic response for aparticular patient, composition, and mode of administration. Theselected dosage level depends upon the activity of the particularcompound, the route of administration, the severity of the conditionbeing treated, and the condition and prior medical history of thepatient being treated. Doses are determined for each particular caseusing standard methods in accordance with factors unique to the patient,including age, weight, general state of health, and other factors thatcan influence the efficacy of the compound(s) of the disclosure. Ingeneral, in the case of oral administration, the THPP compound accordingto the present disclosure or a pharmaceutically acceptable salt thereofis administered at a dose of approximately 30 μg to 100 μg, a dose of 30μg to 500 μg, a dose of 30 μg to 10 g, a dose of 100 μg to 5 g, or adose of 100 μg to 1 g per adult per day. In the case of administrationvia injection, it is administered at a dose of approximately 30 μg to 1g, a dose of 100 μg to 500 mg, or a dose of 100 μg to 300 mg per adultper day. In both cases, a dose is administered once or divided overseveral administrations. Dosage may be simulated, for example, using theSimcyp® program.

It is not intended that the administration of a compound of thedisclosure to a mammal, including humans, be limited to a particularmode of administration, dosage, or frequency of dosing. The presentdisclosure contemplates all modes of administration, including oral,intraperitoneal, intramuscular, intravenous, intraarticular,intralesional, subcutaneous, or any other route sufficient to provide adose adequate to prevent or treat SLE or lupus nephritis. One or morecompounds of the disclosure may be administered to a mammal in a singledose or multiple doses. When multiple doses are administered, the dosesmay be separated from one another by, for example, several hours, oneday, one week, one month, or one year. It is to be understood that, forany particular subject, specific dosage regimes should be adjusted overtime according to the individual need and the professional judgment ofthe person administering or supervising the administration of apharmaceutical composition that includes a compound of the disclosure.

For clinical applications, a compound of the present disclosure maygenerally be administered intravenously, subcutaneously,intramuscularly, colonically, nasally, intraperitoneally, rectally,buccally, or orally. Compositions containing at least one compound ofthe disclosure that is suitable for use in human or veterinary medicinemay be presented in forms permitting administration by a suitable route.These compositions may be prepared according to the customary methods,using one or more pharmaceutically acceptable adjuvants or excipients.The adjuvants comprise, inter alia, diluents, sterile aqueous media, andvarious non-toxic organic solvents. Acceptable carriers or diluents fortherapeutic use are well known in the pharmaceutical field, and aredescribed, for example, in Remington: The Science and Practice ofPharmacy (20th ed.), ed. A. R. Gennaro, Lippincott Williams & Wilkins,2000, Philadelphia, and Encyclopedia of Pharmaceutical Technology, eds.J. Swarbrick and J. C. Boylan, 1988, 1999, Marcel Dekker, New York. Thecompositions may be presented in the form of tablets, pills, granules,powders, aqueous solutions or suspensions, injectable solutions,elixirs, or syrups, and the compositions may optionally contain one ormore agents chosen from the group comprising sweeteners, flavorings,colorings, and stabilizers to obtain pharmaceutically acceptablepreparations.

The choice of vehicle and the content of active substance in the vehicleare generally determined in accordance with the solubility and chemicalproperties of the product, the particular mode of administration, andthe provisions to be observed in pharmaceutical practice. For example,excipients such as lactose, sodium citrate, calcium carbonate, anddicalcium phosphate and disintegrating agents such as starch, alginicacids, and certain complex silicates combined with lubricants (e.g.,magnesium stearate, sodium lauryl sulfate, and talc) may be used forpreparing tablets. To prepare a capsule, it is advantageous to uselactose and high molecular weight polyethylene glycols. When aqueoussuspensions are used, they may contain emulsifying agents thatfacilitate suspension. Diluents such as sucrose, ethanol, polyethyleneglycol, propylene glycol, glycerol, chloroform, or mixtures thereof mayalso be used.

For parenteral administration, emulsions, suspensions, or solutions ofthe compositions of the disclosure in vegetable oil (e.g., sesame oil,groundnut oil, or olive oil), aqueous-organic solutions (e.g., water andpropylene glycol), injectable organic esters (e.g., ethyl oleate), orsterile aqueous solutions of the pharmaceutically acceptable salts areused. The solutions of the salts of the compositions of the disclosureare especially useful for administration by intramuscular orsubcutaneous injection. Aqueous solutions that include solutions of thesalts in pure distilled water may be used for intravenous administrationwith the proviso that (i) their pH is adjusted suitably, (ii) they areappropriately buffered and rendered isotonic with a sufficient quantityof glucose or sodium chloride, and (iii) they are sterilized by heating,irradiation, or microfiltration. Suitable compositions containing acompound of the disclosure may be dissolved or suspended in a suitablecarrier for use in a nebulizer or a suspension or solution aerosol, ormay be absorbed or adsorbed onto a suitable solid carrier for use in adry powder inhaler. Solid compositions for rectal administration includesuppositories formulated in accordance with known methods and containingat least one compound of the disclosure.

Dosage formulations of a compound of the disclosure to be used fortherapeutic administration should be sterile. Sterility is readilyaccomplished by filtration through sterile membranes (e.g., 0.2 micronmembranes) or by other conventional methods. Formulations typically arestored in lyophilized form or as an aqueous solution. The pH of thecompositions of this disclosure in some embodiments, for example, may bebetween 3 and 11, may be between 5 and 9, or may be between 7 and 8,inclusive.

While one route of administration is by oral dosage administration,other methods of administration may be used. For example, compositionsmay be administered subcutaneously, intravenously, intramuscularly,colonically, rectally, nasally, or intraperitoneally in a variety ofdosage forms such as suppositories, implanted pellets or smallcylinders, aerosols, oral dosage formulations, and topical formulationssuch as ointments, drops, and dermal patches. Compounds of embodimentsof the disclosure may be incorporated into shaped articles such asimplants, including but not limited to valves, stents, tubing, andprostheses, which may employ inert materials such as synthetic polymersor silicones, (e.g., Silastic® compositions, silicone rubber, or othercommercially available polymers). Such polymers can includepolyvinylpyrrolidone, pyran copolymer,polyhydroxy-propyl-methacrylamide-phenol,polyhydroxyethyl-aspartamide-phenol, or polyethyleneoxide-polylysinesubstituted with palmitoyl residues. Furthermore, a compound of thedisclosure may be coupled to a class of biodegradable polymers useful inachieving controlled release of a drug, for example polylactic acid,polyglycolic acid, copolymers of polylactic and polyglycolic acid,polyepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters,polyacetals, polydihydropyrans, polycyanoacrylates, and cross linked oramphipathic block copolymers of hydrogels.

A compound of the disclosure may also be administered in the form ofliposome delivery systems, such as small unilamellar vesicles, largeunilamellar vesicles, and multilamellar vesicles. Liposomes can beformed from a variety of lipids, such as cholesterol, stearylamine, orphosphatidylcholines. A compound of the disclosure may also be deliveredusing antibodies, antibody fragments, growth factors, hormones, or othertargeting moieties to which the compound molecules are coupled (e.g.,see Remington: The Science and Practice of Pharmacy, vide supra),including in vivo conjugation to blood components of a compound of anembodiment of the disclosure.

III. Synthesis

General and specific synthesis routes are provided that we found usefulfor preparation of embodiments of the disclosure. Those skilled in theart may recognize that certain variations or modifications of theseprocedures could also lead to synthesis of compounds according to thedisclosure. In some situations the phrase “such as” is used to enumeratevarious alternatives for more generic compounds or structures. It willbe understood that “such as” should not be construed to be limiting, andthat its meaning is in accord with “including, for example, but notlimited to.”

Certain conditions were common to specific examples presented below.Microwave heating was done using a Biotage® Emrys Liberator or Initiatormicrowave reactor. Column chromatography was carried out usingBiotage®SP4 flash chromatography system. Solvent removal was carried outusing either a Büchii rotary evaporator or a Genevac® centrifugalevaporator. NMR spectra were recorded at 400 MHz on a Varian Unity®spectrometer using deuterated solvents. Chemical shifts are reportedrelative to residual protonated solvent.

Thin layer chromatography was performed on Whatman® glass platesprecoated with a 0.25 mm layer of silica gel using various ratios of oneor more of the following solvents: ethyl acetate, heptane,dichloromethane or methanol.

Analytical LC/MS was performed on a Waters Acquity™ system using anXBridge™ C18 1.7 μm 2.1×50 mm column. Solvents A and B are Water w/0.1%formic acid and Acetonitrile w/0.1% formic acid, respectively. 5 minutetotal method time with 5% B to 99% B over 4 minutes with a flow rate of0.3 ml/min. Mass spectral data were acquired on a Waters SQD from100-2000 amu in electrospray positive mode. These conditions arereferred to below as “Condition I.”

Alternatively, purity and mass confirmation were carried out on a WatersAutopurification system using an XBridge™ C8 3.5 μm 4.6×50 mm column.Solvents A and B are water w/0.1% formic acid and acetonitrile w/0.1%formic acid, respectively. 6 minute total method time with 10% B to 95%B over 5 minutes with a flow rate of 2.5 ml/min. Mass spectral data wereacquired on a Micromass ZQ™ from 130-1000 amu in electrospray positivemode. These conditions are referred to below as “Condition II.”

Preparative reverse phase LC/MS was carried out on a WatersAutopurification system using an XBridge™ C8 5 μm, 19×100 mm column.Solvents A and B are water w/0.1% formic acid and Acetonitrile w/0.1%formic acid, respectively. 12 minute total method time with 30% B to 95%B over 10 minutes with a flow rate of 20 ml/min. Mass spectral data wereacquired on a Micromass ZQ™ from 130-1000 amu in electrospray positivemode. These conditions are referred to below as “Condition III.”

Preparative HPLC resolution of racemic compounds was carried out usingone of the following chiral columns: Chiralpak® IA (5 cm×50 cm or 2cm×25 cm), Chiralpak® AD (2 cm×25 cm) or Chiralcel® OD (2 cm×25 cm).Enantiomer ratios of purified compounds were determined by HPLC analysison a 0.45 cm×25 cm column comprised of the same stationary phase (IA, ADor OD).

General methods and experimentals for preparing compounds of the presentdisclosure are set forth below. In certain cases, a particular compoundis described by way of example. However, it will be appreciated that ineach case a series of compounds of the present disclosure were preparedin accordance with the schemes and experimentals described below. Forthose compounds where NMR and/or mass spectrometry data are available,the data is presented immediately following the description of thesynthesis of the compound or in Table 11.

The following abbreviations are used herein:

DEFINITIONS

The following abbreviations have the indicated meanings:

HATU: N,N,N′,N′-Tetramethyl-O-(7-azabenzotriazol-1-yl)uroniumhexafluorophosphate

DIEA: N,N-diisopropylethylamine

DMAP: 4-Dimethylaminopyridine

TEA: triethylamine

DMF: N,N-dimethylformamide

NMP: N-methylpyrrolidine

THF: tetrahydrofuran

DCM: dichloromethane

MTBE: methyl tert-butyl ether

TFA: trifluoroacetic acid

EDC: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride

NaOH: sodium hydroxide

NaBH4: sodium borohydride

IPA: isopropyl alcohol or isopropanol

EtOH: ethanol

EtOAc: ethyl acetate

TLR: Toll-like receptor

DAMP: Danger-Associated Molecular Pattern

PAMP: Pathogen-Associated Molecular Pattern

IFN: interferon

pDC: plasmacytoid dendritic cell

PBMC: peripheral blood mononuclear cell

qPCR: quantitative polymerase chain reaction

TLDA: Taqman® Low Density Array

PBS: phosphate buffered saline

ssRNA: single-stranded RNA

dsDNA: double-stranded DNA

SOC: standard-of-care

R848: resiquimod

HCQ: hydroxychloroquine

HCl: hydrochloric acid

aq.: aqueous

AcOH: acetic acid

PhNTf₂: N-phenyltrifluoromethanesulfonimide

Tf: trifluoromethanesulfonate

MeOH: methanol

ee: enantiomeric excess

HEPES: 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid

NH₄Cl: ammonium chloride

Example 1 General Synthetic Methods

Compounds of the disclosure were made according to the general syntheticscheme shown below:

The preparation of several of the examples use the route depicted inScheme 1. Typically, a commercially available nitrile, such as aromatic,heterocyclic, aliphatic or heteroaliphatic nitrile (I) is subjected toacetonitrile in presence of a strong protic organic base such aspotassium t-amylate in a neutral solvent such as toluene at 0° C. orroom temperature to provide the cyanoimine (II). The resulting imine isthen reacted with hydrazine in the presence of an organic acid such asacetic acid to provide the key intermediate or 3-substituted-5-aminopyrrazole (III). The right half of the these examples is prepared by wayof a Claisen condensation using an aromatic or heteroaromatic acylketone(V) with a substituted acetylester such as trifluoro, difluoro ornonsubstituted acetyl ester (IV) to form the diketone (VI).

Condensation of the two intermediates III and VI provides the2,3-b-pyrazolopyrimidine in the presence of a mild organic acid such asacetic acid. In some cases the 5-substituted-3-amino pyrrazoles (III)are commercially available and are used in this reaction directly.Reduction to form the racemic 5,7-cis tetrahydropyrazolopyrimidine(VIII) is effected using a hydride source or by catalytic hydrogenation.Resolution via chiral, high performance liquid chromatography or usingdiastereomeric crystallization provides the final desired products IXand X. In many examples the racemic mixture of compound VIII wereevaluated biologically without further purification via chiralresolution.

Scheme 2 exemplifies an alternative route to various compounds of thedisclosure starting with commercially available3-hydroxy-5-aminopyrazole condensed with diketone VI to provide2-hydroxypyrazolopyrimidine X as a key intermediate. Treatment of X witha suitable trifluoromethylsulfonylation reagent provides triflateintermediate XI. Reduction of triflate XI, followed by Suzukicross-coupling with a variety of boronates, where R¹ is an aromatic,heteroaromatic, allylic, heteroallylic or aliphatic gives the racemicfinal product VIII. Alternatively, triflate XI is subjected to Suzukicross-coupling first, and the intermediate compound XIV is then reducedto the racemic final product VIII. As in Scheme 1, racemic compound VIIIis resolved into its constituent enantiomers by way of chiralchromatography or by diastereomeric crystallization.

The route used in Scheme 3 is a modification of Scheme 2 in which abromide replaces the triflate, i.e. the triflate group in compound XI isreplaced by bromide as shown in XVI. 3-bromo-5-aminopyrazole wassynthesized according to literature methods (Moy, et al. J. Med. Chem.2010, 53, 1238). The conditions for the Suzuki cross-coupling areessentially the same as those in Scheme 2.

The route in Scheme 4 reverses the Suzuki cross-coupling startingmaterials by the generation of the boronate ester on the keypyrazolopyrimidine followed condensation of a suitable electrophile(XIX, X═OTf, Cl, Br, I) to generate VIII after reduction.

Scheme 5 depicts the final steps in the synthesis of several of theexamples of general structure XV presented herein. Accordingly, thecommon intermediate used is benzonitrile XX, typically substituted atthe 3- or 4-position with respect to the pyrazolopyrimidine moiety,which is converted to benzoic acid XXI via acidic or basic hydrolysis.Coupling of XXI with a suitable amine (R⁴R⁵NH) provides the racemicamide XXII, which is resolved into its pure enantiomer XV.Alternatively, the chiral resolution can be carried out either at thestage of the benzonitrile (XX→XXIII) or the benzoic acid (XXI→XXIV) andthe enantiopure materials transformed in a similar manner to the finalproduct XV.

Synthetic Examples Section A Example 1 Synthesis of ER-892887

To a 5-L flask charged with terephthalonitrile (300.0 g, 2.34 mol) wasadded toluene (1.80 L, followed by acetonitrile (245 ml) at roomtemperature. 24.2 wt % Potassium-t-amylate (1.672 L, 1.338 kg, 2.58 mol)was added while controlling temperature to <30° C. over a 1-hour period.

The mixture (yellow thick paste) was allowed to cool to 20° C. andstirred for 16 hours, after which time water (0.9 L) was added to thevigorously stirred mixture until yellow solids became light tan solids.The mixture was filtered through a glass filter, the resulting solid wasrinsed with water (1.8 L) followed by IPA (1.8 L), collected and driedunder vacuum at 40° C. for 46 h. 374.1 g of Compound A-2 (2.21 mol, 94%yield) was obtained as light tan powder.

¹H NMR (400 MHz, METHANOL-d₄) δ ppm 4.26 (s, 1H) 7.72-7.77 (m, 2H)7.77-7.82 (m, 2H).

MS (M+H⁺) 170.1.

To a 5-L flask was charged with 1-(3,4-dimethoxyphenyl)ethanone (487 g,2.70 mol) was added THF (487 mL) followed by MTBE (1.46 L). Ethyltrifluoroacetate (516 mL, 4.33 mol) was added followed by 25 wt %solution of sodium methoxide (701 g, 3.24 mol) in methanol at roomtemperature. The resultant mixture was warmed up to 40° C. and stirredat 40-43° C. for 16 h after which time the reaction mixture was cooledto 10° C. and poured into a pre-cooled (10° C.) mixture of MTBE (2.44 L)and 20 wt % citric acid (1.217 g, 1.279 mol) while maintainingT-internal <20° C. After 30 min vigorous stirring, the organic layer wasseparated and sequentially washed with two times with 20 wt % sodiumchloride (1.46 L) and then concentrated to approximately ⅓^(rd) thevolume.

The resultant residue was diluted with MTBE (3.90 L), washed with water(1.95 L) and concentrated to approximately ⅓^(rd) the volume upon whichtime a significant amount of product solids precipitated from thesolution. The resultant mixture was azeotroped two times dryness withn-heptane (1.95 L). n-Heptane (877 mL) and MTBE (97 mL) were added andresultant mixture was heated to 50° C., allowed to cool to 15° C. over a2-h period and then precipitate was filtered. The resulting tan solidwas dried under vacuum oven (40° C. for 16 h then 20° C. for 48 h) toprovide 714.3 g of Compound A-6 (2.59 mol, 96% yield).

¹H NMR (400 MHz, CHLOROFORM-d) δ ppm 3.97 (s, 3H) 3.98 (s, 3H) 6.53 (s,1H) 6.94 (d, J=8.51 Hz, 1H) 7.48 (d, J=2.10 Hz, 1H) 7.60 (dd, J=8.51,2.10 Hz, 1H).

MS (M+H⁺) 277.2.

Compound A-2 (250 g, 1.478 mol) was suspended in acetic acid (1.0 L)with stirring at room temperature after which time hydrazine (72.5 mL,1.478 mol) in water (75 mL) was added with the internal temperatureincreasing to 42° C. The resultant suspension was heated to 70° C. andmaintained at that temperature for 5 min at which time the suspensionturned to almost a clear mixture and then became cloudy again to providecompound A-3 in situ.

Toluene (1.50 L) was quickly added to the above mixture (temperaturedropped to 48° C.) followed by compound A-6 (408 g, 1.478 mol). Theresultant mixture was heated to 80° C. upon which time a significantamount of yellow solids accumulated generating very thick paste. Aftermaintain the reaction at 80° C. for 30 min, the mixture was cooled to50° C. and iced water (1.50 L) was added with vigorous stirring. Theyellow precipitate was collected by filtration and washed with water(3.75 L), water (2.5 L), and IPA (2.50 L). The resultant yellow solidwas dried under vacuum (40° C.) for 3 days to provide 464 g of CompoundA-7 (1.09 mol, 74% yield).

¹H NMR (400 MHz, METHANOL-d₄) δ ppm 3.90 (s, 4H) 3.95 (s, 4H) 7.10 (d,J=8.54 Hz, 1H) 7.30 (s, 1H) 7.83 (d, J=8.01 Hz, 2H) 7.89 (d, J=2.02 Hz,1H) 7.92 (s, 1H) 8.23 (d, J=8.66 Hz, 2H).

MS (M+H⁺) 425.4.

Compound A-7 (326 g, 768.2 mmol) was suspended in ethanol (3.26 L) andwater (815 ml) followed by heating to 68-70° C. A sodium borohydridestock solution [previously prepared separately by adding sodiumborohydride (96 g, 2.54 mol) to an aqueous solution of 0.1M sodiumhydroxide (815 ml, 81.50 mmol) at room temperature followed by stirringfor 30 min] was added at room temperature over 1.5 hours upon which thereaction temperature rose to 68-71° C. Upon complete addition themixture was heated at 68-71° C. for 4 h after which time the mixture wascooled to 40° C. and acetone (564 mL) was added over 30 min (T-internal40-42° C.) followed by stirring an additional 30 min at 40-42° C. toprovide compound A-8 in situ.

¹H NMR (400 MHz, CHLOROFORM-d) δ ppm 2.33-2.44 (m, 1H) 2.51 (d, J=6.09Hz, 1H) 3.91 (s, 3H) 3.93 (s, 3H) 4.33 (s, 1H) 4.45 (dd, J=11.80, 2.08Hz, 1H) 4.87-4.97 (m, 1H) 5.81 (s, 1H) 6.87-6.91 (m, 1H) 6.96-7.01 (m,2H) 7.64-7.68 (m, 2H) 7.85-7.90 (m, 2H).

MS (M+H⁺) 428.9.

Sodium hydroxide (461 g, 11.52 mol) was added to the above mixture afterwhich time the exothermic reaction was heated to 70-73° C. and stirredfor 16 hours. After cooling to room temperature, ice water (3.260 L) wasadded to the mixture to cool it to 10° C. followed by a slow addition of10% aqueous HCl (4.75 L) while maintaining the temperature to <25° C.with vigorous stirring. Vigorous stirring continued for 30 min. Thefinal reaction mixture was decanted over a glass filter to recover anybrown crude product within the solvent.

The solid that remained in the reaction vessel was suspended in water(3.26 L) and stirred vigorously for 30 min and decanted as above. Thisprocess was repeated an additional time. The final recovered solids wererinsed with water (3.26 L) followed by IPA (2.61 L) and dried underair/vacuum for 1 hand then under vacuum at 45° C. for 20 h to provide226.7 g of Compound A-9 (0.507 mol, 66% yield) as a light tan solid.

¹H NMR (400 MHz, DMSO-d₆) δ ppm 3.76 (s, 3H) 3.79 (s, 3H) 5.86 (s, 1H)6.84 (s, 1H) 6.92-7.04 (m, 2H) 7.08 (d, J=1.50 Hz, 1H) 7.84 (m, J=8.33Hz, 2H) 7.95 (m, J=8.23 Hz, 2H).

MS (M+H⁺) 448.4.

Compound A-9 (333.8 g, 746.1 mmol) was dissolved in DMF (1.335 L) andthen diluted with DCM (1.00 L) while stirring at room temperature.Tert-butyl piperazine-1-carboxylate (139 g, 746.1 mmol) was addedfollowed by stirring at room temperature for an additional 20 min. EDC(143 g, 746.1 mmol) was added the resultant mixture was stirred for 2hours at room temperature. n-Heptane (4.01 L) was added to thevigorously stirred mixture followed by water (5.34 L) while maintainingthe temperature below 25° C. The resultant biphasic mixture was stirredat room temperature for an additional 30 min after which time the solidswere filtered, followed by washing with water (4.0 L) and then n-heptane(2.0 L). The solid was dried under vacuum at 50° C. for 18 h to provide478.5 g of crude compound A-12.

473 g of crude Compound A-12 was suspended in a solvent mixture ofn-heptane (1.0 L) and IPA (1.0 L) followed by heating to 50° C. andstirred at 50° C. for an additional 10 min. The suspension was cooled to20° C. over a 30-min period followed by stirring for an additional 30min. The solid was filtered, washed with solvent mixture of IPA (500 mL)and n-heptane (500 mL) and then dried in under vacuum at 50° C. for 5 h.This above suspension and filtration process was repeated one additionaltime. 403 g of Compound A-12 (0.655 mol, 88% yield?) was obtained as atan powder.

¹H NMR (400 MHz, METHANOL-4) δ ppm 0.94-1.14 (m, 1H) 1.45-1.51 (m, 9H)1.57-1.75 (m, 1H) 2.17-2.29 (m, 1H) 2.49-2.58 (m, 1H) 3.84 (s, 3H) 3.87(s, 3H) 4.45 (d, J=11.44 Hz, 1H) 5.03-5.15 (m, 1H) 6.96-7.01 (m, 1H)7.03-7.08 (m, 1H) 7.11 (d, J=1.79 Hz, 1H) 7.40-7.52 (m, 1H) 7.85 (d,J=8.09 Hz, 2H) 7.99-8.08 (m, 1H).

MS (M+H⁺) 616.3

Compound A-12 (340 g, 0.552 mol) was suspended in DCM (510 ml, 7.926 moland TFA (510 ml, 6.619 mol) was added to the suspension over 10 min. Themixture was stirred at 23-27° C. for 3 h, after which the mixture wascooled to 15° C. and diluted with water (1700 ml, 9.436 mol) whilekeeping the internal temperature below 20° C. The mixture was dilutedwith n-heptane (3.4 L, 23.209 mol) then cooled to 15° C. Sodiumhydroxide (2.872 Kg, 7.179 mol) was added over 30 min while controllingT-internal <25° C. The resultant mixture was stirred at 20-25° C. for 20min then filtered to collect light tan precipitates. The precipitate wasfirst rinsed with water (3.4 L) and then a mixture of n-heptane (1.36 L)and DCM (204 ml). The wet cake was transferred to a tray and dried invacuum oven at 50° C. overnight to afford 248.8 g (0.483 mol, 87% yield)of ER-890044 as a light tan solid.

ER-890044 (30.3 g, 58.8 mmol) was dissolved in a 1:1 solution ofdichloromethane and methanol (120 mL) and the cloudy solution filteredthrough a medium porosity Buchner funnel. The clear filtrate wasdirectly used for chiral HPLC purification. 4 mL of this solution wasloaded onto a 5 cm×50 cm Chiralpak IA column and eluted with a mobilephase comprising of heptane, methanol and ethanol (4:3:3 ratio; modifiedwith 0.06% diethylamine) at a flow rate of 75 mL/min. ER-892887 wascollected between 18.5 min and 23.2 min, while ER-892924 was collectedbetween 24.2 min and 32 min. 30 such injections were carried out and thepooled fractions concentrated under reduced pressure to provide the pureenantiomeric products ER-892887 (10.5 g, 20.3 mmol, >95% ee) andER-892924 (9.8 g, 19.0 mmol, >95% ee).

To a solution of ER-892887 (8.50 g, 16.5 mmol) in dry ethyl acetate (500mL), was added 4 M HCl in Dioxane (4.53 mL, 18.1 mmol) drop wise over 10min. A white precipitate was obtained. The resulting suspension wasstirred at ambient temperature for 15 min, then filtered under reducedpressure. The collected solid was washed with ethyl acetate (2×100 mL)and ether (2×100 mL) and dried under vacuum to afford 8.29 g ofER-892887 hydrochloride salt (15.0 mmol, 91% yield)) as a white solid.

ER-892930

To a 5 mL screw-cap reaction tube was added compound A-94-(7-(difluoromethyl)-5-(3,4-dimethoxyphenyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidin-2-yl)benzoicacid (21.7 mg, 0.049 mmol), 3-Amino-1,2-propanediol (13.2 mg, 0.145mmol), HATU (1.10 eq, 24.8 mg, 0.065 mmol), NMP (0.500 ml), and Hünig'sBase (6.00 ul, 0.044 mmol). The reaction mixture was stirred at 30° C.overnight. The material was purified by LC/MS using HPLC condition III.The fractions containing product were combined and concentrated in vacuoto give compound ER-892930 as an off-white solid (13.3 mg, 53% yield).

ER-894463

To a 5 mL screw-cap reaction tube was added compound A-94-(7-(difluoromethyl)-5-(3,4-dimethoxyphenyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidin-2-yl)benzoicacid (30.0 mg, 0.067 mmol), tert-butyl 3-aminopyrrolidine-1-carboxylate(74.9 mg, 0.402 mmol), HATU (50.9 mg, 0.134 mmol), NMP (0.500 ml), andHünig's Base (6.00 ul, 0.044 mmol). The reaction mixture was stirred at30° C. overnight. The material was purified by LC/MS using HPLCcondition III. The fractions containing product were combined andconcentrated in vacuo. The resulting material was then taken up inethanol (1.0 ml) and 4.0 M HCl in Dioxane (1.0 ml, 4.00 mmol) and themixture stirred at RT for 1 h. The mixture was then concentrated invacuo to give compound ER-894463 as a yellow solid (22.1 mg, 60% yield).

ER-895080

To a 5 mL screw-cap reaction tube was added compound A-94-(7-(difluoromethyl)-5-(3,4-dimethoxyphenyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidin-2-yl)benzoicacid (20.0 mg, 0.045 mmol), tert-butyl2,6-diazaspiro[3.4]octane-6-carboxylate (18.98 mg, 0.089 mmol), HATU(18.7 mg, 0.049 mmol), NMP (0.500 ml), and Hünig's Base (6.00 ul, 0.044mmol). The reaction mixture was stirred at 30° C. overnight. Thematerial was purified by LC/MS using HPLC condition III. The fractionscontaining product were combined and concentrated in vacuo. Theresulting material was then taken up in ethanol (1.0 ml) and 50%trifluoroacetic acid in DCM (1.0 ml, 4.00 mmol) and the mixture stirredat RT for 1 h. The mixture was then concentrated in vacuo to givecompound ER-895080 as a yellow solid (2.40 mg, 8% yield).

Example ER-894462-00 was prepared from compound ER-886619 (30.0 mg,0.067 mmol) and commercially available azetidin-3-ol (29.4 mg, 0.402mmol) in a manner similar to that of example ER-895080. Purification byLCMS, using HPLC condition III, afforded the desired product (8.1 mg,24%).

Example ER-894465-00 was prepared from compound ER-886619 (30.0 mg,0.067 mmol) and commercially available pyrrolidin-3-ol (35.0 mg, 0.402mmol) in a manner similar to that of example ER-895080. Purification byLCMS, using HPLC condition III, afforded the desired product (18.5 mg,53%).

Example ER-894464-01 was prepared from compound ER-886619 (30.0 mg,0.067 mmol) and commercially available tert-butylpyrrolidin-3-ylcarbamate (74.9 mg, 0.402 mmol) in a manner similar tothat of example ER-894463. Purification by LCMS, using HPLC conditionIII, afforded the desired product (32.6 mg, 88%).

Example ER-895077-01 was prepared from compound ER-886619 (30.0 mg,0.067 mmol) and commercially available tert-butyl3-aminoazetidine-1-carboxylate (69.3 mg, 0.402 mmol) in a manner similarto that of example ER-894463. Purification by LCMS, using HPLC conditionIII, afforded the desired product (8.6 mg, 24%).

Example ER-895078-01 was prepared from compound ER-886619 (30.0 mg,0.067 mmol) and commercially available tert-butyl azetidin-3-ylcarbamate(69.3 mg, 0.402 mmol) in a manner similar to that of example ER-894463.Purification by LCMS, using HPLC condition III, afforded the desiredproduct (29.7 mg, 82%).

Example ER-895746-01 was prepared from compound ER-886619 (7.8 mg, 0.062mmol) and commercially available tert-butylhexahydropyrrolo[3,4-c]pyrrole-2(1H)-carboxylate (37.0 mg, 0.174 mmol)in a manner similar to that of example ER-894463. Purification by LCMS,using HPLC condition III, afforded the desired product (27.5 mg, 77%).

Example ER-895748-01 was prepared from compound ER-886619 (25.0 mg,0.056 mmol) and commercially available (S)-tert-butyl3-aminopiperidine-1-carboxylate (35.5 mg, 0.177 mmol) in a mannersimilar to that of example ER-894463. Purification by LCMS, using HPLCcondition III, afforded the desired product (31.3 mg, 99%).

Example ER-895749-01 was prepared from compound ER-886619 (25.0 mg,0.056 mmol) and commercially available (R)-tert-butyl3-aminopiperidine-1-carboxylate (36.6 mg, 0.183 mmol) in a mannersimilar to that of example ER-894463. Purification by LCMS, using HPLCcondition III, afforded the desired product (29.5 mg, 93%).

Example ER-895750-01 was prepared from compound ER-886619 (25.0 mg,0.056 mmol) and commercially available (S)-tert-butyl3-methylpiperazine-1-carboxylate (31.7 mg, 0.158 mmol) in a mannersimilar to that of example ER-894463. Purification by LCMS, using HPLCcondition III, afforded the desired product (30.6 mg, 97%).

Example ER-895751-01 was prepared from compound ER-886619 (25.0 mg,0.056 mmol) and commercially available (R)-tert-butyl3-methylpiperazine-1-carboxylate (43.5 mg, 0.217 mmol) in a mannersimilar to that of example ER-894463. Purification by LCMS, using HPLCcondition III, afforded the desired product (31.2 mg, 98%).

Example ER-895752-01 was prepared from compound ER-886619 (25.0 mg,0.056 mmol) and commercially available (2S,5R)-tert-butyl2,5-dimethylpiperazine-1-carboxylate (30.3 mg, 0.141 mmol) in a mannersimilar to that of example ER-894463. Purification by LCMS, using HPLCcondition III, afforded the desired product (30.6 mg, 94%).

Example ER-895753-01 was prepared from compound ER-886619 (25.0 mg,0.056 mmol) and commercially available (2R,5R)-tert-butyl2,5-dimethylpiperazine-1-carboxylate (37.6 mg, 0.175 mmol) in a mannersimilar to that of example ER-894463. Purification by LCMS, using HPLCcondition III, afforded the desired product (19.5 mg, 60%).

Example ER-895754-01 was prepared from compound ER-886619 (25.0 mg,0.056 mmol) and commercially available (1S,4S)-tert-butyl2,5-diazabicyclo[2.2.1]heptane-2-carboxylate (33.5 mg, 0.169 mmol) in amanner similar to that of example ER-894463. Purification by LCMS, usingHPLC condition III, afforded the desired product (26.7 mg, 85%).

Example ER-895083-15 was prepared from compound ER-886619 (24.5 mg,0.055 mmol) and commercially available tert-butyl(azetidin-3-ylmethyl)(methyl)carbamate (22.4 mg, 0.112 mmol) in a mannersimilar to that of example ER-895080. Purification by LCMS, using HPLCcondition III, afforded the desired product (0.6 mg, 2%).

Example ER-895081-15 was prepared from compound ER-886619 (20.6 mg,0.046 mmol) and commercially available tert-butyl(6-aminospiro[3.3]heptan-2-yl)carbamate (23.4 mg, 0.103 mmol) in amanner similar to that of example ER-895080. Purification by LCMS, usingHPLC condition III, afforded the desired product (13.3 mg, 43%).

Example ER-895082-15 was prepared from compound ER-886619 (23.5 mg,0.053 mmol) and commercially available tert-butyl2,7-diazaspiro[4.4]nonane-2-carboxylate (25.1 mg, 0.111 mmol) in amanner similar to that of example ER-895080. Purification by LCMS, usingHPLC condition III, afforded the desired product (5.3 mg, 15%).

Example ER-898416 was prepared.3-(5-(3,4-dimethoxyphenyl)-7-(trifluoromethyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidin-2-yl)benzoicacid was prepared from (3-cyanophenyl)boronic acid (93 mg, 0.631 mmol)and5-(3,4-dimethoxyphenyl)-7-(trifluoromethyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidin-2-yltrifluoromethanesulfonate (100.0 mg, 0.21 mmol) and in a manner similarto that of5-(3,4-dimethoxyphenyl)-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-7-(trifluoromethyl)pyrazolo[1,5-a]pyrimidine(section G, preparation of ER893393) to yield3-(5-(3,4-dimethoxyphenyl)-7-(trifluoromethyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidin-2-yl)benzonitrile(46 mg, 51% yield). Hydrolysis of material (75 mg, 0.175 mmol) thusprepared in a manner similar to that of compound A-9 afforded thedesired compound (42 mg, 54% yield)

Example ER-895811 was prepared in two steps from3-(5-(3,4-dimethoxyphenyl)-7-(trifluoromethyl)-4,5,6,7-tetrahydropyrazolo[1,5a]-pyrimidin-2-yl)benzoicacid (24 mg, 0.054 mmol) and commercially available (S)-tert-butyl3-aminopyrrolidine-1-carboxylate (40 mg, 0.215 mmol) in a manner similarto that of example D-6 to afford intermediate (3S)-tert-butyl3-(3-(5-(3,4-dimethoxyphenyl)-7-(trifluoromethyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidin-2-yl)benzamido)pyrrolidine-1-carboxylate(7 mg, 0.011 mmol, 21% yield). Then, this intermediate (6 mg, 0.0097mmol) was treated with HCl in a manner similar to that of exampleER-897560 to afford the desired product ER-895811 (4.4 mg, 82% yield).

Example ER-896386 was prepared in two steps from3-(5-(3,4-dimethoxyphenyl)-7-(trifluoromethyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidin-2-yl)benzoicacid (119 mg, 0.266 mmol) and commercially available tert-butylhexahydropyrrolo[3,4-c]pyrrole-2(1H)-carboxylate (226 mg, 1.06 mmol) ina manner similar to that of example D-6 to afford intermediatetert-butyl5-(3-(5-(3,4-dimethoxyphenyl)-7-(trifluoromethyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidin-2-yl)benzoyl)hexahydropyrrolo[3,4-c]pyrrole-2(1H)-carboxylate(94 mg, 0.146 mmol, 55% yield). Then, this intermediate was resolvedinto its constituent enantiomers in a manner similar to that ofER-890044 (Section A) to afford tert-butyl5-(3-((5S,7R)-5-(3,4-dimethoxyphenyl)-7-(trifluoromethyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidin-2-yl)benzoyphexahydropyrrolo[3,4-c]pyrrole-2(1H)-carboxylate(33 mg, 0.051 mmol) and tert-butyl5-(3-((5R,7S)-5-(3,4-dimethoxyphenyl)-7-(trifluoromethyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidin-2-yl)benzoyl)hexahydropyrrolo[3,4-c]pyrrole-2(1H)-carboxylate(33 mg, 0.051 mmol). Then, intermediate tert-butyl5-(34(5S,7R)-5-(3,4-dimethoxyphenyl)-7-(trifluoromethyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidin-2-yebenzoyehexahydropyrrolo[3,4-c]pyrrole-2(1H)-carboxylate(22 mg, 0.034 mmol) was treated with HCl in a manner similar to that ofexample ER-897560 to afford the desired product ER-896386 (19 mg, 96%yield).

Example ER-896387 was prepared from intermediate tert-butyl5-(3-((5S,7R)-5-(3,4-dimethoxyphenyl)-7-(trifluoromethyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidin-2-yl)benzoyl)hexahydropyrrolo[3,4-c]pyrrole-2(1H)-carboxylateobtained in example ER-896386. Then, this intermediate (16 mg, 0.025mmol) was treated with HCl in a manner similar to that of exampleER-897560 to afford the desired product ER-896387 (14 mg, 99% yield).

Example ER-896388 was prepared in two steps from3-(5-(3,4-dimethoxyphenyl)-7-(trifluoromethyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidin-2-yl)benzoicacid (25 mg, 0.056 mmol) and commercially available tert-butylpiperazine-1-carboxylate (42 mg, 0.224 mmol) in a manner similar to thatof example D-6 to afford intermediate tert-butyl4-(3-(5-(3,4-dimethoxyphenyl)-7-(trifluoromethyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidin-2-yl)benzoyl)piperazine-1-carboxylate(18 mg, 0.029 mmol, 52% yield). Then, this intermediate (16 mg, 0.026mmol) was treated with HCl in a manner similar to that of exampleER-897560 to afford the desired product ER-896388 (14 mg, 98% yield).

Example ER-896389 was prepared in two steps from compound3-(5-(3,4-dimethoxyphenyl)-7-(trifluoromethyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidin-2-yl)benzoicacid (25 mg, 0.056 mmol) and commercially availabletert-butyl((3R,5S)-5-methylpiperidin-3-yl)carbamate (48 mg, 0.224 mmol)in a manner similar to that of example D-6 to afford intermediatetert-butyl((3R,5S)-1-(3-(5-(3,4-dimethoxyphenyl)-7-(trifluoromethyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidin-2-yl)benzoyl)-5-methylpiperidin-3-yl)carbamate(22 mg, 0.034 mmol, 61% yield). Then, this intermediate (20 mg, 0.031mmol) was treated with HCl in a manner similar to that of exampleER-897560 to afford the desired product ER-896389 (17.5 mg, 97% yield).

Synthetic Examples Section B Example 2 Preparation of ER-885681

Compound B-1 was prepared from commercially available tert-butyl4-cyanopiperidine-1-carboxylate in manner similar to compound A-8 (56 g,70% yield for the final step).

¹H NMR (400 MHz, CHLOROFORM-d) δ ppm 1.44-1.49 (m, 10H) 1.50-1.62 (m,6H) 1.89 (d, J=12.05 Hz, 2H) 2.31 (dt, J=13.69, 11.51 Hz, 1H) 2.41-2.49(m, 1H) 2.69-2.88 (m, 3H) 3.89 (s, 3H) 3.91 (s, 3H) 4.06-4.21 (m, 3H)4.38 (dd, J=11.71, 1.95 Hz, 1H) 4.81 (dt, J=11.29, 5.80 Hz, 1H) 5.28 (s,1H) 6.84-6.89 (m, 1H) 6.93-6.98 (m, 2H).

MS (M+H⁺) 511.0.

Trifluoroacetic Acid (15 mL) was added to a solution of compound B-1(7.86 g, 15.4 mmol) in Methylene chloride (15 mL) and the mixturestirred at rt. After 15 min, the reaction mixture was concentrated underreduced pressure, and the residue partitioned between MTBE and sat.NaHCO₃. The layers were separated and the aq. solution further extractedwith dichloromethane (2×). The combined extracts were washed with brine,dried (Na2SO4) and concentrated under reduced pressure to affordER-887084 as a white solid (4.28 g. 68%).

¹H NMR (400 MHz, METHANOL-d₄) δ ppm 1.52-1.64 (m, 3H) 1.84 (br. s., 1H)2.06-2.17 (m, 1H) 2.40-2.47 (m, 1H) 2.59-2.70 (m, 5H) 3.01-3.09 (m, 3H)3.79 (s, 3H) 3.82 (s, 3H) 4.33 (dd, J=11.59, 2.25 Hz, 1H) 4.87-4.95 (m,1H) 5.30 (s, 1H) 6.93 (d, J=8.31 Hz, 1H) 6.96-7.00 (m, 1H) 7.04 (d,J=1.90 Hz, 1H).

MS (M+H⁺) 411.5.

Compound ER-887084 (1.19 g, 2.91 mmol) was dissolved in methanol (11 mL)and the solution further diluted with 19 mL of 1:1 ethanol-heptanesolution modified with 0.05% diethylamine. 1 mL of this solution wasloaded onto a 2 cm×25 cm Chiralcel OD column and eluted with a mobilephase comprising of 1:1 heptane-ethanol (containing 0.05% diethylamine)at a flow rate of 15 mL/min. Compound ER-885681 was collected between7.3 min and 9.6 min, while compound ER-885682 was collected between 9.8min and 14.5 min. 33 such injections were carried out and the pooledfractions concentrated under reduced pressure to provide the pureenantiomeric products compound ER-885681 (571 mg, 96% yield, >95% ee)and compound ER-885682 (ER-887275) (574 mg, 96% yield, >95% ee).

Examples ER-885454 and ER-885455

Compound ER-880894 was prepared from commercially available4-cyanopyridine (450 g, 4.32 mol) using the general sequence outlined inScheme I and exemplified by the preparation of compound A-8 (ER-890044)to yield ER-880894 (205 g, mmol 12% overall yield).

¹H NMR (400 MHz, CHLOROFORM-d) δ ppm 2.29-2.46 (m, 2H) 2.47-2.60 (m, 2H)3.91 (s, 3H) 3.93 (s, 3H) 4.36 (s, 1H) 4.45 (d, J=9.96 Hz, 1H) 4.93 (dt,J=11.28, 5.79 Hz, 2H) 5.85 (s, 1H) 6.85-6.93 (m, 1H) 6.93-7.04 (m, 2H)7.59-7.70 (m, 2H) 8.56-8.66 (m, 2H).

MS (M+H⁺) 405.2.

Resolution of ER-880894 (200 mg, 0.495 mmol) by chiral HPLC in a mannersimilar to that of ER-890044 (with the exception that 1:1ethanol-heptane was used as the mobile phase) gave ER-885454 (96 mg, 48%yield) and ER-885455 (91.2 mg, 46% yield).

Examples ER-886622 and ER-886623

Example ER-886434 was prepared from commercially available4-cyano-2-methoxypyridine (mg, mmol) in a manner similar to that ofcompound ER-880894 to yield ER-886434 (127 mg, 62% yield)

MS (M+H+) 435.6.

Resolution by chiral HPLC in a manner similar to that of ER-880894 gaveER-886622 (21.5 mg, 44% yield, >95% ee) and ER-886623 (20.2 mg, 41%yield, >95% ee).

Synthetic Examples Section C Preparation of ER-890035

A solution of compound C-1 (9.91 g, 100 mmol) and compound C-2 (27.6 g,100 mmol) in acetic acid (60 mL) was heated at 80° C. The clear darkorange reaction mixture turned progressively lighter as a yellowprecipitate began to form. Within 30 min, the reaction mixture hadturned into a thick yellow slurry. The mixture maintained at 80° C. foranother 15 min. The mixture was cooled to rt, and IPA added. Thesuspension was heated to approximately 75° C., then cooled to roomtemperature. The yellow precipitate was filtered, and the solids washedwith additional IPA, and dried under reduced pressure. Compound C-3 wasobtained as a light yellow solid (30.9 g, 91% yield).

¹H NMR (400 MHz, METHANOL-d₄) d ppm 3.92-3.92 (m, 3H) 3.96 (s, 3H) 7.10(d, J=8.51 Hz, 1H) 7.67 (s, 1H) 7.75 (dd, J=8.47, 2.21 Hz, 1H) 7.83-7.86(in, 1H) (major tautomer).

MS (M+H⁺) 340.5.

Compound C-4

Compound C-3 (9.9 g, 29.2 mmol) was dissolved inN,N-Diisopropylethylamine (25.4 mL) and 1,2-dichloroethane (100 mL) andthe mixture stirred at rt. To this solution was addedN-phenylbis(trifluoromethanesulphonimide) (20.8 g, 58.2 mmol) and themixture heated at reflux. After 45 min, the reaction mixture was cooledto rt, and concentrated under reduced pressure to afford an orange brownresidue. The residue was purified by silica gel chromatography usinggradient elution (10 to 40% EtOAc in Heptane) to afford compound C-4 asa yellow solid (7.10 g, 52% yield). A small amount of the N-sulfonylatedregioisomer was formed in the reaction, but not isolated.

¹H NMR (400 MHz, DMSO-d₆) δ ppm 3.87 (s, 3H) 3.90 (s, 3H) 7.15 (d,J=8.65 Hz, 1H) 7.20 (s, 1H) 7.85 (d, J=2.14 Hz, 1H) 8.00 (dd, J=8.55,2.14 Hz, 1H) 8.35 (s, 1H).

MS (M+H⁺) 472.3.

Compound C-5

To a solution of compound C-4 (2.24 g, 0.00475 mol) in Ethanol (29.9mL), was added sodium borohydride (360 mg, 9.50 mol) and the mixturestirred at rt. After 30 min, the reaction mixture was carefully pouredinto a 1:1 mixture of saturated NH₄Cl solution and water. The aqueoussolution was extracted with dichloromethane (3×); the combined extractswashed with brine, dried and concentrated under reduced pressure. Thecrude product was purified by silica gel chromatography using gradientelution (0 to 50% EtOAc in heptane) to afford compound C-5 as a whitesolid (1.94 g, 86% yield).

¹H NMR (400 MHz, METHANOL-d₄) δ ppm 2.18 (dt, J=13.35, 11.43 Hz, 1H)2.47 (ddd, J=13.43, 5.95, 2.59 Hz, 1H) 3.80 (s, 3H) 3.82 (s, 3H) 4.44(dd, J=11.63, 2.44 Hz, 1H) 4.92-5.03 (m, 1H) 5.32 (s, 1H) 6.94 (d,J=8.28 Hz, 1H) 6.99 (dd, J=8.41, 1.89 Hz, 1H) 7.04 (d, J=1.95 Hz, 1H).MS (M+H⁺) 472.3.

Compounds C-6 and C-7

Compound C-5 (900 mg, 1.91 mmol) was dissolved in methanol (10 mL) andsolution further diluted with ethanol (5 mL) and heptane (5 mL). 1 mL ofthis solution was loaded onto a 2 cm×25 cm Chiralcel® OD column andeluted with a mobile phase comprising of 1:1 heptane-ethanol at a flowrate of 15 mL/min. Compound C-6 (ER-887274) was collected between 6.0min and 7.7 min, while compound C-7 (ER-887275) was collected between8.75 min and 11.5 min. 17 such injections were carried out and thepooled fractions concentrated under reduced pressure to provide the pureenantiomeric products compound C-6 (ER-887274) (683 mg, 1.44 mmol, >95%ee) and compound C-7 (ER-887275) (671 mg, 1.41 mmol, >95% ee).

Example ER-890035. To a 15 mm×75 mm screw cap tube was added(3-(5-methyl-1,2,4-oxadiazol-3-yl)phenyl)boronic acid (36.1 mg, 0.177mmol), 0.0750 M of Tetrakis(triphenylphosphine)palladium(0) in1,4-Dioxane (80.0 uL, 0.006 mmol), 0.150 M5-(3,4-dimethoxyphenyl)-7-(trifluoromethyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidin-2-yltrifluoromethanesulfonate (C-5) in 1,4-dioxane (420.0 uL, 0.0631 mmol),and 2.00 M of sodium carbonate in water (60.0 uL, 120 mmol). Thereaction vessel was purged with nitrogen and sealed. The vial was shakenand heated in an aluminum block at 85° C. overnight. To the mixture wasadded 1.0 mL of saturated aqueous sodium bicarbonate and the mixture wasthen extracted with ethyl acetate (2×2.0 mL). The combined organiclayers were concentrated in vacuo. The remaining residue was dissolvedin 500 μL of DMSO. The material was purified by LC/MS on an XTerra C819×100 mm column with an acetonitrile:water (formic acid) gradient. Thefractions containing product were combined and concentrated in vacuo togive compound ER-890035 as a yellow solid, 2.9 mg (9.5% yield).

Example ER-893972 was prepared from compound C-6 (250 mg, 0.526 mmol)and commercially available (1,4-dimethyl-1H-indazol-5-yl)boronic acid(200 mg, 1.05 mmol) in a manner similar to that of example ER-890035.Purification by silica gel chromatography afforded the desired product(159 mg, 64% yield).

Example ER-892893 was prepared from compound C-6 (250 mg, 0.526 mmol)and commercially available (4-((3-hydroxypropyl)carbamoyl)phenyl)boronicacid (235 mg, 1.05 mmol) in a manner similar to that of exampleER-890035. Purification by silica gel chromatography afforded thedesired product (166 mg, 63% yield).

Example ER-892892 was prepared from compound C-6 (250 mg, 0.526 mmol)and commercially available (4-carbamoyl-2-fluorophenyl)boronic acid (192mg, 1.05 mmol) in a manner similar to that of example ER-890035.Purification by silica gel chromatography afforded the desired product(179 mg, 73% yield).

Example ER-892892 was prepared from compound C-6 (250 mg, 0.526 mmol)and commercially available (4-carbamoyl-2-fluorophenyl)boronic acid (192mg, 1.05 mmol) in a manner similar to that of example ER-890035.Purification by silica gel chromatography afforded the desired product(179 mg, 73% yield). Example ER-894680 was prepared from compound C-6(250 mg, 0.526 mmol) and commercially available(4-methyl-1H-indazol-5-yl)boronic acid (185 mg, 1.05 mmol in a mannersimilar to that of example ER-890035. Purification by silica gelchromatography afforded the desired product (90 mg, 37% yield).

Example ER-887734 was prepared from compound C-6 (20 mg, 0.042 mmol) andcommercially available (3-acetamidophenyl)boronic acid (22.6 mg, 0.126mmol) in a manner similar to that of example ER-890035. Purification byLCMS (Rt 7.45 min, condition II) afforded the desired product (9.7 mg,50% yield).

Example ER-887738 was prepared from compound C-6 (20 mg, 0.042 mmol) andcommercially available (3-acetamidophenyl)boronic acid (20.3 mg, 0.126mmol) in a manner similar to that of example ER-890035. Purification byLCMS (Rt 8.58 min, condition II) afforded the desired product (6.5 mg,35% yield).

Example ER-892889 was prepared from compound C-6 (200 mg, 0.421 mmol)and commercially available (3-chloro-4-(methylcarbamoyephenyl)boronicacid (180 mg, 0.843 mmol in a manner similar to that of exampleER-890035. Purification by silica gel chromatography afforded thedesired product (88 mg, 42% yield).

Example ER-892890 was prepared from compound C-6 (250 mg, 0.526 mmol)and commercially available (4-carbamoyl-3-chlorophenyl)boronic acid (210mg, 1.05 mmol in a manner similar to that of example ER-890035.Purification by silica gel chromatography afforded the desired product(138 mg, 55% yield).

Example ER-893961 was prepared from compound C-6 (40 mg, 0.084 mmol) andcommercially available (1,6-dimethyl-1H-indazol-5-yl)boronic acid (51mg, 0.268 mmol in a manner similar to that of example ER-890035.Purification by LCMS afforded the desired product (8.9 mg, 22% yield).

Example ER-893961 was prepared from compound C-6 (40 mg, 0.084 mmol) andcommercially available5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-benzo[d]imidazol-2(3H)-one(80.55 mg, 0.309 mmol in a manner similar to that of example ER-890035.Purification by LCMS afforded the desired product (4.3 mg, 11% yield).

Example ER-889871 was prepared from compound C-5 (50.0 mg, 0.105 mmol)and commercially available (4-carbamoylphenyl)boronic acid (2.50 eq) ina manner similar to that of example ER-890035. Purification by LCMS,using HPLC condition III, afforded the desired product (12.8 mg, 27%).

Example ER-889874 was prepared from compound C-5 (50.0 mg, 0.105 mmol)and commercially available (4-(hydroxymethyl)phenyl)boronic acid (2.50eq) in a manner similar to that of example ER-890035. Purification byLCMS, using HPLC condition III, afforded the desired product (7.8 mg,17%).

Example ER-890017 was prepared from compound C-5 (30.0 mg, 0.063 mmol)and commercially available (4-carbamoyl-2-fluorophenyl)boronic acid(2.50 eq) in a manner similar to that of example ER-890035. Purificationby LCMS, using HPLC condition III, afforded the desired product (13.4mg, 46%).

Example ER-890019 was prepared from compound C-5 (30.0 mg, 0.063 mmol)and commercially available (4-carbamoyl-3-fluorophenyl)boronic acid(2.50 eq) in a manner similar to that of example ER-890035. Purificationby LCMS, using HPLC condition III, afforded the desired product (11.7mg, 40%).

Example ER-890020 was prepared from compound C-5 (30.0 mg, 0.063 mmol)and commercially available (4-carbamoyl-3-chlorophenyl)boronic acid(2.50 eq) in a manner similar to that of example ER-890035. Purificationby LCMS, using HPLC condition III, afforded the desired product (14 mg,46%).

Example ER-890024 was prepared from compound C-5 (30.0 mg, 0.063 mmol)and commercially available (4-((3-hydroxypropyl)carbamoyl)phenyl)boronicacid (2.50 eq) in a manner similar to that of example ER-890035.Purification by LCMS, using HPLC condition III, afforded the desiredproduct (11.5 mg, 36%).

Example ER-890027 was prepared from compound C-5 (30.0 mg, 0.063 mmol)and commercially available (4-(benzylcarbamoyl)-3-chlorophenyl)boronicacid (2.50 eq) in a manner similar to that of example ER-890035.Purification by LCMS, using HPLC condition III, afforded the desiredproduct (12.3 mg, 34%).

Example ER-890028 was prepared from compound C-5 (30.0 mg, 0.063 mmol)and commercially available(3-chloro-4-(isopropylcarbamoyl)phenyl)boronic acid (2.50 eq) in amanner similar to that of example ER-890035. Purification by LCMS, usingHPLC condition III, afforded the desired product (18.1 mg, 55%).

Example ER-890029 was prepared from compound C-5 (30.0 mg, 0.063 mmol)and commercially available (3-chloro-4-(methylcarbamoyl)phenyl)boronicacid (2.50 eq) in a manner similar to that of example ER-890035.Purification by LCMS, using HPLC condition III, afforded the desiredproduct (15.4 mg, 49%).

Example ER-890035 was prepared from compound C-5 (30.0 mg, 0.063 mmol)and commercially available(3-(5-methyl-1,2,4-oxadiazol-3-yl)phenyl)boronic acid (2.50 eq) in amanner similar to that of example ER-890035. Purification by LCMS, usingHPLC condition III, afforded the desired product (2.9 mg, 9%).

Example ER-890043 was prepared from compound C-5 (30.0 mg, 0.063 mmol)and commercially available(2-oxo-2,3-dihydro-1H-benzo[d]imidazol-5-yl)boronic acid (2.50 eq) in amanner similar to that of example ER-890035. Purification by LCMS, usingHPLC condition III, afforded the desired product (5.6 mg, 19%).

Example ER-890044 was prepared from compound C-5 (30.0 mg, 0.063 mmol)and commercially available (4-(piperazine-1-carbonyl)phenyl)boronic acid(2.50 eq) in a manner similar to that of example ER-890035. Purificationby LCMS, using HPLC condition III, afforded the desired product (13.7mg, 42%).

Example ER-890050-00 was prepared from compound C-5 (30.0 mg, 0.063mmol) and commercially available (3-(1H-pyrazol-1-yl)phenyl)boronic acid(2.50 eq) in a manner similar to that of example ER-890035. Purificationby LCMS, using HPLC condition III, afforded the desired product (15.4mg, 52%).

Example ER-891029-00 was prepared from compound C-5 (30.0 mg, 0.063mmol) and commercially available(6-(methylcarbamoyl)pyridin-3-yl)boronic acid (2.50 eq) in a mannersimilar to that of example ER-890035. Purification by LCMS, using HPLCcondition III, afforded the desired product (9 mg, 31%).

Example ER-891043 was prepared from compound C-5 (30.0 mg, 0.063 mmol)and commercially available (1,7-dimethyl-1H-indazol-5-yl)boronic acid(2.50 eq) in a manner similar to that of example ER-890035. Purificationby LCMS, using HPLC condition III, afforded the desired product (18 mg,61%).

Example ER-891044 was prepared from compound C-5 (30.0 mg, 0.063 mmol)and commercially available (1,4-dimethyl-1H-indazol-5-yl)boronic acid(2.50 eq) in a manner similar to that of example ER-890035. Purificationby LCMS, using HPLC condition III, afforded the desired product (14.3mg, 48%).

Example ER-891047 was prepared from compound C-5 (30.0 mg, 0.063 mmol)and commercially available (1,6-dimethyl-1H-indazol-5-yl)boronic acid(2.50 eq) in a manner similar to that of example ER-890035. Purificationby LCMS, using HPLC condition III, afforded the desired product (18.1mg, 61%).

Example ER-891058 was prepared from compound C-5 (30.0 mg, 0.063 mmol)and commercially available(4-(cyclopropylcarbamoyl)-3-fluorophenyl)boronic acid (2.50 eq) in amanner similar to that of example ER-890035. Purification by LCMS, usingHPLC condition III, afforded the desired product (13.8 mg, 43%).

Example ER-892908 was prepared from compound C-5(30.0 mg, 0.063 mmol)and commercially available(4-(tert-butylcarbamoyl)-3-fluorophenyl)boronic acid (2.50 eq) in amanner similar to that of example ER-890035. Purification by LCMS, usingHPLC condition III, afforded the desired product (7 mg, 21%).

Example ER-892931 was prepared from compound C-5 (32.6 mg, 0.069 mmol)and commercially available (1H-indazol-4-yl)boronic acid (2.70 eq) in amanner similar to that of example ER-890035. Purification by LCMS, usingHPLC condition III, afforded the desired product (5.5 mg, 18%).

Section C.1 (Compounds Prepared by Cross-Coupling from Bromide)

A mixture of 3-amino-5-bromopyrazole C-8 (3.24 g, 20 mmol) and diketoneC-2 (5.52 g, 20 mmol) in Acetic acid (20 mL) was heated to 80° C. After1 h, the reaction mixture was cooled to rt, and diluted with IPA. Ayellow ppt formed in the mixture, which was collected by filtration.Upon standing, additional ppt was formed in the mother liquor. Thecombined crops were collected and dried under vacuum to afford 3.89 g ofcompound C-9 as a yellow solid (9.67 mmol, 48% yield).

¹H NMR (400 MHz, CHLOROFORM-d) δ ppm 3.99 (s, 3H) 4.05 (s, 3H) 6.85 (s,1H) 7.00 (d, J=8.47 Hz, 1H) 7.57 (s, 1H) 7.62 (dd, J=8.45, 2.16 Hz, 1H)7.80 (d, J=2.14 Hz, 1H)

MS (M+H⁺) 402.2

Compound C-10

Compound C-9 (1.1 g, 2.74 mmol) was suspended in ethanol (5 mL) andsodium borohydride (155 mg, 4.1 mmol) added. The mixture was stirred atrt for 1.5 h. At this time additional Sodium borohydride (155 mg) wasadded and the mixture stirred at rt for another 2 h. Acetic acid (1.56mL) was added to quench the reaction and the resulting aq solutionpoured into sat. NaHCO3 solution. The precipitated white solid wascollected by filtration, washed with water, and dried under reducedpressure to afford 667 mg of compound C-10 as an off-white solid (1.64g, 60% yield).

¹H NMR (400 MHz, CHLOROFORM-d) δ ppm 2.34-2.37 (m, 1H) 2.49 (m, 1H) 3.86(s, 1H) 3.91 (s, 3H) 3.92 (s, 3H) 4.40 (dd, J=11.75, 2.44 Hz, 1H) 4.82(dt, J=11.32, 5.73 Hz, 1H) 5.51 (s, 1H) 6.80-6.91 (m, 2H) 6.93-7.00 (m,1H)

MS (M+H⁺) 406.4

Example ER-889925 was prepared from compound C-10 (37.0 mg, 0.091 mmol)and commercially available isoquinolin-5-ylboronic acid (2.50 eq) in amanner similar to that of example ER-890035. Purification by LCMS, usingHPLC condition III, afforded the desired product (9.9 mg, 24%).

Synthetic Examples Section D

5-(5-3,4-dimethoxyphenyl)-7-(trifluoromethyl)pyrazolo[1,5-a]pyrimidin-2-yl)picolinonitrile(D-3)

To a vial was added5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)picolinonitrile (1.95 g,8.48 mmol), dicyclohexyl(2′,6′-dimethoxy-[1,1′-biphenyl]-2-yl)phosphine(0.261 g, 0.636 mmol),5-(3,4-dimethoxyphenyl)-7-(trifluoromethyl)pyrazolo[1,5-a]pyrimidin-2-yltrifluoromethanesulfonate (2.00 g, 4.24 mmol), potassium acetate (0.833g, 8.49 mmol) and palladium(II) acetate (0.095 g, 0.424 mmol). Afterflushing vial and contents with nitrogen, a degassed solution of 1:1toluene/ethanol (20 mL) was added, vial was sealed and heated to 80° C.for 2 days. Ethyl acetate was added to the resulting light yellowprecipitate was collected by filtration and dried under high vacuum(1.13 g, 63%). This material was used in the next step without furtherpurification.

¹H NMR (400 MHz CDCl₃) δ ppm 4.00 (3H), 4.06 (3H), 7.02 (2H), 7.21 (1H),7.66 (2H), 7.84 (2H), 8.5 (1H), 9.33 (1H).

MS (M+H⁺) 426.0.

5-(5-(3,4-dimethoxyphenyl)-7-(trifluoromethyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidin-2-yl)picolinonitrile(D-4)

To a mixture of5-(5-3,4-dimethoxyphenyl)-7-(trifluoromethyl)pyrazolo[1,5-a]pyrimidin-2-yl)picolinonitrile(1.03 g, 2.43 mmol) in ethanol (23.0 mL) was added sodium borohydride(0.276 g, 7.29 mmol); reaction mixture was heated to reflux for 2 hafter which the reaction mixture was allowed to cool to room temperatureand stir overnight. Acetic acid (1.40 mL) followed by 1.0 N hydrochloricacid solution (48.6 mL) was added and the mixture was concentrated usingrotary evaporation. Dichloromethane was added to the residue andfiltered. Concentration of the filtrate by rotary evaporation affordedcrude product as a light brown foam (0.694 g, 66%). This material wasused in the next step without further purification.

¹H NMR (400 MHz DMSO-d6) δ ppm 2.13 (1H), 2.46 (1H), 3.77 (6H), 4.48(1H), 5.36 (1H), 5.97 (1H), 6.98 (3H), 7.08 (1H), 8.08 (1H), 8.28 (1H),9.05 (1H).

MS (M+H⁺) 430.0.

5-(5-(3,4-dimethoxyphenyl)-7-(trifluoromethyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidin-2-yl)picolinicacid (D-5)

To a suspension of5-(5-(3,4-dimethoxyphenyl)-7-(trifluoromethyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidin-2-yl)picolinonitrile(0.694 g, 1.62 mmol) was added 4.00 M sodium hydroxide solution in water(2.02 mL). The resulting mixture was heated at reflux for 3 h. 1.0 Nhydrochloric acid solution (11.3 mL) was added and the light brownprecipitate was filtered, rinsed with copious amounts of water, driedunder air/vacuum for 30 min, and then under high vacuum 48 h to give alight brown solid (0.535 g, 74%). This material was used in the nextstep without further purification.

¹H NMR (400 MHz DMSO-d6) δ ppm 2.13 (1H), 2.46 (1H), 3.76 (3H), 3.79(3H), 4.48 (1H), 5.35 (1H), 6.00 (1H), 6.99 (3H), 7.08 (1H), 8.11 (1H),8.32 (1H), 9.04 (1H).

MS (M+H⁺) 449.01

Tert-butyl4-(5-(5-(3,4-dimethoxyphenyl)-7-(trifluoromethyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidin-2-yl)picolinoyl)piperazine-1-carboxylate(D-6)

To a solution of5-(5-(3,4-dimethoxyphenyl)-7-(trifluoromethyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidin-2-yl)picolinicacid (458 mg, 1.02 mmol) in DMF (4.0 mL) was addedN,N-diisopropylethylamine (535 μl, 3.06 mmol) and HATU (427 mg, 1.12mmol). After stirring 1 h at rt, tert-butyl piperazine-1-carboxylate(209 mg, 1.12 mmol) was added and the reaction was stirred overnight atroom temperature. Water was added and the resulting light brownprecipitate was collected by filtration (0.5385 g). Purification bycolumn chromatography (15% to 100% ethyl acetate/heptane affordedtert-butyl4-(5-(5-(3,4-dimethoxyphenyl)-7-(trifluoromethyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidin-2-yl)picolinoyepiperazine-1-carboxylate(332 mg, 54%) as a light yellow solid.

¹H NMR (400 MHz CD3OD) δ ppm 1.46 (9H), 2.2 (1H), 2.5 (1H), 3.50 (8H),3.73 (1H), 3.82 (6H), 4.45 (1H), 5.08 (1H), 5.91 (1H), 7.02 (3H), 7.62(1H), 8.23 (1H), 8.92 (1H).

MS (M+H⁺) 617.2.

(5-(5-(3,4-Dimethoxyphenyl)-7-(trifluoromethyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidin-2-yl)pyridin-2-yl)(piperazin-1-yl)methanone(ER-897560)

Compound D-6 (66 mg, 0.107 mmol) was resolved into its constituentenantiomers in a manner similar to that of ER-890044 (Section A) toafford tert-butyl4-(5-((5S,7R)-5-(3,4-dimethoxyphenyl)-7-(trifluoromethyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidin-2-yl)picolinoyl)piperazine-1-carboxylate(9.2 mg, 14% yield) and tert-butyl4-(5-((5R,7S)-5-(3,4-dimethoxyphenyl)-7-(trifluoromethyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidin-2-yl)picolinoyl)piperazine-1-carboxylate(9.2 mg, 14% yield).

To a solution of tert-butyl4-(5-((5S,7R)-5-(3,4-dimethoxyphenyl)-7-(trifluoromethyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidin-2-yl)picolinoyl)piperazine-1-carboxylate(90.0 mg, 0.146 mmol) in ethanol (4 mL) was added 4.0 M HCl in1,4-dioxane (1.84 mL). The reaction was heated to 40° C. for 1 h. Thereaction mixture was concentrated by rotary evaporation and azeotropedwith toluene to give ER-897560 as a pale yellow solid (91 mg, 106%yield).

Example ER-897097 was prepared from compound C-9 (500 mg, 1.243 mmol)and commercially available tert-butyl4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-5,6-dihydropyridine-1(2H)-carboxylate(461 mg, 1.492 mmol) in a manner similar to that of example D-4.Purification by silica gel chromatography afforded the Boc-protectedER-897097 (460 mg, 73% yield). Deprotection of this Boc-protectedproduct (100 mg, 0.197 mmol) was carried in a manner similar to that ofER-887084 (Section B) to afford ER-897097 (80 mg, 99% yield). (LC-MS: Rt1.49 min, (M+1)⁺409.08 under condition II)

Example ER-897269

Compound ER-897097 (94 mg, 0.229 mmol) was resolved into its constituentenantiomers in a manner similar to that of ER-890044 (Section A) toafford one of the isomers ER-897269 (15 mg, 16% yield, >95% ee).

Example ER-897105 was prepared from compound C-9 (500 mg, 1.243 mmol)and commercially available tert-butyl4-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazol-1-yl)piperidine-1-carboxylate(563 mg, 1.492 mmol) in a manner similar to that of example D-4.Purification by silica gel chromatography afforded the Boc-protectedER-897105 (312 mg, 44% yield). Deprotection of this Boc-protectedproduct (100 mg, 0.173 mmol) was carried in a manner similar to that ofER-887084 (Section B) to afford ER-897105 (71 mg, 86% yield).

LC-MS: Rt 1.48 min, (M+1)⁺477.09 under condition II.

Examples ER-897214 and ER-897215

Compound ER-897105 (50 mg, 0.105 mmol) was resolved into its constituentenantiomers in a manner similar to that of ER-890044 (Section A) toafford ER-897214 (19 mg, 39% yield, >95% ee) and ER-897215 (19 mg, 39%yield, >95% ee).

Example ER-897381 was prepared from compound C-4 (500 mg, 1.061 mmol)and commercially available tert-butyl4-(5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyrimidin-2-yl)piperazine-1-carboxylate(621 mg, 1.591 mmol) in a manner similar to that of example D-4.Purification by silica gel chromatography afforded the Boc-protectedER-897381 (311 mg, 50% yield). Deprotection of this Boc-protectedproduct (100 mg, 0.17 mmol) was carried in a manner similar to that ofER-887084 (Section B) to afford ER-897381 (75 mg, 90% yield). LC-MS: Rt1.52 min, (M+1)⁺489.21 under condition II).

Example ER-897714 and ER-897715

Compound ER-897381 (71 mg, 0.134 mmol) was resolved into its constituentenantiomers in a manner similar to that of ER-890044 (Section A) toafford ER-897714 (27 mg, 38% yield, >95% ee) and ER-897716 (30 mg, 42%yield, >95% ee).

Example ER-897405 was prepared from compound C-4 (500 mg, 1.061 mmol)and commercially available tert-butyl4-(5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-2-yl)piperazine-1-carboxylate(619 mg, 1.591 mmol) in a manner similar to that of example D-4.Purification by silica gel chromatography afforded the Boc-protectedER-897405 (311 mg, 65% yield). Deprotection of this Boc-protectedproduct (100 mg, 0.17 mmol) was carried in a manner similar to that ofER-887084 (Section B) to afford ER-897405 (72 mg, 87% yield). LC-MS: Rt1.45 min, (M+1)⁺489.04 under condition II.

Example ER-897716 and ER-897717

Compound ER-897405 (70 mg, 0.143 mmol) was resolved into its constituentenantiomers in a manner similar to that of ER-890044 (Section A) toafford ER-897716 (23 mg, 33% yield) and ER-897717 (23 mg, 33% yield).

Example ER-897765 was prepared from compound C-4 (438 mg, 0.93 mmol) andcommercially availabletert-butyl(1-(5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-2-yl)piperidin-4-yl)carbamate(250 mg, 0.62 mmol) in a manner similar to that of example D-4.Purification by silica gel chromatography afforded the Boc-protectedER-897765 (161 mg, 43% yield). Deprotection of this Boc-protectedproduct (161 mg, 0.267 mmol) was carried in a manner similar to that ofER-887084 (Section B) to afford ER-897765 (131 mg, 98% yield).

Example ER-895809 was prepared in two steps from compound D-5 (19 mg,0.042 mmol) and commercially available (S)-tert-butyl3-aminopyrrolidine-1-carboxylate (32 mg, 0.169 mmol) in a manner similarto that of example D-6 to afford intermediate (3S)-tert-butyl3-(5-(5-(3,4-dimethoxyphenyl)-7-(trifluoromethyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidin-2-yl)picolinamido)pyrrolidine-1-carboxylate(19 mg, 0.031 mmol, 72% yield). Then, this intermediate (17 mg, 0.028mmol) was treated with HCl in a manner similar to that of exampleER-897560 to afford the desired product ER-895809 (16 mg, 98% yield).

Example ER-895810 was prepared in two steps from compound D-5 (19 mg,0.042 mmol) and commercially available (R)-tert-butyl3-aminopyrrolidine-1-carboxylate (32 mg, 0.169 mmol) in a manner similarto that of example D-6 to afford intermediate (3R)-tert-butyl3-(5-(5-(3,4-dimethoxyphenyl)-7-(trifluoromethyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidin-2-yl)picolinamido)pyrrolidine-1-carboxylate(24 mg, 0.039 mmol, 92% yield). Then, this intermediate (22 mg, 0.036mmol) was treated with HCl in a manner similar to that of exampleER-897560 to afford the desired product ER-895810 (21 mg, 100% yield).Exact mass calculated: 516.21. Observed: 517.5.

Example ER-896133 was prepared in two steps from compound D-5 (15 mg,0.033 mmol) and commercially available tert-butylpiperazine-1-carboxylate (25 mg, 0.134 mmol) in a manner similar to thatof example D-6 to afford intermediate tert-butyl4-(5-(5-(3,4-dimethoxyphenyl)-7-(trifluoromethyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidin-2-yl)picolinoyl)piperazine-1-carboxylate(6 mg, 0.009 mmol, 29% yield). Then, this intermediate (6 mg, 0.009mmol) was treated with HCl in a manner similar to that of exampleER-897560 to afford the desired product ER-896133 (5.3 mg, 92% yield).

Example ER-896134 was prepared in two steps from compound D-5 (15 mg,0.033 mmol) and commercially available tert-butyl4-aminopiperidine-1-carboxylate (27 mg, 0.134 mmol) in a manner similarto that of example D-6 to afford intermediate tert-butyl4-(5-(5-(3,4-dimethoxyphenyl)-7-(trifluoromethyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidin-2-yl)picolinamido)piperidine-1-carboxylate(7 mg, 0.011 mmol, 33% yield). Then, this intermediate (7 mg, 0.011mmol) was treated with HCl in a manner similar to that of exampleER-897560 to afford the desired product ER-896134 (5.8 mg, 87% yield).

Example ER-896135 was prepared in two steps from compound D-5 (15 mg,0.033 mmol) and commercially available (S)-tert-butyl3-aminopiperidine-1-carboxylate (27 mg, 0.134 mmol) in a manner similarto that of example D-6 to afford intermediate (3S)-tert-butyl3-(5-(5-(3,4-dimethoxyphenyl)-7-(trifluoromethyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidin-2-yl)picolinamido)piperidine-1-carboxylate(7 mg, 0.011 mmol, 33% yield). Then, this intermediate (7 mg, 0.011mmol) was treated with HCl in a manner similar to that of exampleER-897560 to afford the desired product ER-896135 (6.5 mg, 97% yield).

Example ER-896136 was prepared in two steps from compound D-5 (15 mg,0.033 mmol) and commercially available (R)-tert-butyl3-aminopiperidine-1-carboxylate (27 mg, 0.134 mmol) in a manner similarto that of example D-6 to afford intermediate (3R)-tert-butyl3-(5-(5-(3,4-dimethoxyphenyl)-7-(trifluoromethyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidin-2-yl)picolinamido)piperidine-1-carboxylate(7 mg, 0.011 mmol, 33% yield). Then, this intermediate (7 mg, 0.011mmol) was treated with HCl in a manner similar to that of exampleER-897560 to afford the desired product ER-896136 (6.1 mg, 91% yield).

Example ER-896137 was prepared from compound D-5 (15 mg, 0.033 mmol) andcommercially available propan-2-amine (8 mg, 0.134 mmol) in a mannersimilar to that of example D-6 to afford the desired product ER-896137(5 mg, 31% yield).

4-(5-(3,4-dimethoxyphenyl)-7-(trifluoromethyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidin-2-yl)picolinicacid was prepared in a manner similar to that of example D-5 (101 mg,29% yield).

Example ER-897034 was prepared in two steps from4-(5-(3,4-dimethoxyphenyl)-7-(trifluoromethyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidin-2-yl)picolinicacid (25 mg, 0.056 mmol) and commercially available tert-butylpiperazine-1-carboxylate (31 mg, 0.167 mmol) in a manner similar to thatof example D-6 to afford intermediate tert-butyl4-(4-(5-(3,4-dimethoxyphenyl)-7-(trifluoromethyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidin-2-yl)picolinoyl)piperazine-1-carboxylate(15 mg, 0.024 mmol, 44% yield). Then, this intermediate (13 mg, 0.021mmol) was treated with HCl in a manner similar to that of exampleER-897560 to afford the desired product ER-897034 (12.2 mg, 98% yield).

Example ER-897036 was prepared in two steps from4-(5-(3,4-dimethoxyphenyl)-7-(trifluoromethyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidin-2-yl)picolinicacid (25 mg, 0.056 mmol) and commercially available (S)-tert-butyl3-aminopiperidine-1-carboxylate (33 mg, 0.167 mmol) in a manner similarto that of example D-6 to afford intermediate (3S)-tert-butyl3-(4-(5-(3,4-dimethoxyphenyl)-7-(trifluoromethyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidin-2-yl)picolinamido)piperidine-1-carboxylate(15 mg, 0.024 mmol, 43% yield). Then, this intermediate (13 mg, 0.021mmol) was treated with HCl in a manner similar to that of exampleER-897560 to afford the desired product ER-897036 (12 mg, 96% yield.

Example ER-897037 was prepared in two steps from4-(5-(3,4-dimethoxyphenyl)-7-(trifluoromethyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidin-2-yl)picolinicacid (25 mg, 0.056 mmol) and commercially available tert-butyl4-aminopiperidine-1-carboxylate (33 mg, 0.167 mmol) in a manner similarto that of example D-6 to afford intermediate tert-butyl4-(4-(5-(3,4-dimethoxyphenyl)-7-(trifluoromethyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidin-2-yl)picolinamido)piperidine-1-carboxylate(14 mg, 0.022 mmol, 40% yield). Then, this intermediate (13 mg, 0.021mmol) was treated with HCl in a manner similar to that of exampleER-897560 to afford the desired product ER-897037 (11 mg, 90% yield).

Synthetic Examples Section F

4-(5-amino-1H-pyrazol-3-yl)benzonitrile (compound A-2; 5.31 g, 31.4mmol) and commercially available1-(3,4-dimethoxyphenyl)-4,4-difluorobutane-1,3-dione (8.1 g, 31.4 mmol)in a manner similar to that of compound A-7 afforded compound F-1 (9.84g, 77% yield). MS (M+H⁺) 407.3.

Compound F-2 (9.8 g, 99% yield) was obtained by NaBH₄ reduction of F-1(9.8 g, 24.1 mmol) in a manner similar to that of compound A-8.

¹H NMR (400 MHz, METHANOL-d₄) δ ppm 2.17-2.39 (m, 2H) 3.83-3.85 (m, 3H)3.85-3.88 (m, 3H) 4.47 (dd, J=10.87, 2.29 Hz, 1H) 4.66-4.81 (m, 1H) 5.86(s, 1H) 6.60 (t, J=55.00 Hz, 1H) 6.97 (d, J=7.97 Hz, 1H) 7.01-7.07 (m,1H) 7.10 (s, 1H) 7.66-7.76 (m, 2H) 7.91 (d, J=8.13 Hz, 2H). MS (M+H⁺)411.7.

Compound F-3 (9.4 g, 100% yield) was obtained by hydrolysis of F-1 (8.94g, 21.8 mmol) using aq. NaOH in a manner similar to that of compoundA-9.

¹H NMR (400 MHz, METHANOL-d₄) δ ppm 2.21-2.37 (m, 2H) 3.80-3.82 (m, 4H)3.84 (s, 3H) 4.46 (dd, J=10.83, 2.56 Hz, 1H) 6.60 (t, J=55.00 Hz, 1H)6.95 (d, J=8.28 Hz, 1H) 7.00-7.04 (m, 1H) 7.08 (d, J=1.91 Hz, 1H) 7.81(in, J=8.35 Hz, 2H) 8.00 (in, J=8.39 Hz, 2H). MS (M+H⁺) 431.4.

To a 5 mL screw-cap reaction tube was added compound F-34-(7-(difluoromethyl)-5-(3,4-dimethoxyphenyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidin-2-yl)benzoicacid (1.99 g, 4.64 mmol), tert-butyl piperazine-1-carboxylate (1.05 g,5.61 mmol), HATU (2.14 g, 5.62 mmol), DMF (10.0 ml), and Hünig's Base(0.815 ml, 4.67 mmol). The reaction mixture was stirred at 30° C.overnight. The mixture was diluted with ethyl acetate, washed withwater, washed with 0.1N aqueous hydrochloric acid, washed with aqueoussodium bicarbonate, washed with brine, dried over anhydrous magnesiumsulfate, filtered, and concentrated in vacuo to give a brown solid. Thematerial was purified by flash chromatography with a heptane:ethylacetate gradient 0-90%. The fractions containing product were combinedand concentrated in vacuo to give an off-white solid. The resultantsolid was then taken up in ETHANOL (10.0 ml) and 4.0 M HCl in Dioxane(10.0 ml, 40.00 mmol) and the mixture stirred at RT for 1 h. The mixturewas then concentrated in vacuo to give compound ER-894466 as a yellowsolid (1.91 g, 77% yield).

Chiral resolution of ER-894466 (1.91 g, 3.59 mmol) by HPLC in a mannersimilar to ER-890044 provided ER-895302 (538 mg, 56% yield, >95% ee) andER-895303 (671 mg, 70% yield, >95% ee).

5-amino-1H-pyrazol-3-ol (9.91 g, 100 mmol) and1-(3,4-dimethoxyphenyl)-4,4-difluorobutane-1,3-dione (25.8 g, 100 mmol)in a manner similar to that of compound A-14, gave compound F-47-(difluoromethyl)-5-(3,4-dimethoxyphenyl)pyrazolo[1,5-a]pyrimidin-2-ol(30.6 g, 95 mmol, 95% yield).

¹H NMR (400 MHz, METHANOL-d₄) δ ppm 3.87-3.90 (m, 3H) 3.90-3.93 (m, 3H)5.94 (s, 1H) 7.06 (d, J=8.39 Hz, 1H) 7.29 (t, J=53.00 Hz, 1H) 7.47 (s,1H) 7.67 (dd, J=8.43, 1.45 Hz, 1H) 7.77 (d, J=1.75 Hz, 1H). MS (M+H⁺)323.3.

To a suspension of compound F-47-(difluoromethyl)-5-(3,4-dimethoxyphenyl)pyrazolo[1,5-a]pyrimidin-2-ol(3.21 g, 9.99 mmol) in DMF (5.00 ml) and THF (25.0 ml) at 0° C. wasadded sodium hydride (0.480 g, 11.99 mmol) in small portions. Themixture was stirred at 0° C. for 30 min.1,1,1-Trifluoro-N-phenyl-N-(trifluoromethyl)sulfonyl methanesulfonamide(4.28 g, 11.99 mmol) was added in small portions and the mixture allowedto attain rt. After 1 h, the reaction mixture was poured into 0.1 N HClsolution giving rise to a white ppt, which was filtered and washed withwater. The crude product was suspended in IPA, heated to ˜70° C. andcooled to rt. The white ppt obtained was washed with additional IPA, anddried under reduced pressure to afford compound F-57-(difluoromethyl)-5-(3,4-dimethoxyphenyl)pyrazolo[1,5-a]pyrimidin-2-yltrifluoromethanesulfonate as a white solid (2.85 g, 6.29 mmol, 62.9%yield)

¹H NMR (400 MHz, METHANOL-d₄) δ ppm 3.89 (s, 3H) 3.92 (s, 3H) 6.70 (s,1H) 7.07 (d, J=8.50 Hz, 1H) 7.17-7.27 (m, 2H) 7.29-7.38 (m, 1H) 7.78(dd, J=8.51, 2.14 Hz, 1H) 7.82-7.89 (m, 2H). MS (M+H⁺) 454.4.

Compound F-6 (27.2 g, 77% yield) was obtained by NaBH4 reduction ofcompound F-5 (35.0 g, 77.2 mmol) in a manner similar to that of compoundA-16.

¹H NMR (400 MHz, METHANOL-d₄) δ ppm 2.14-2.32 (m, 2H) 3.79 (s, 3H) 3.81(s, 3H) 4.44 (dd, J=11.06, 2.67 Hz, 1H) 4.53-4.66 (m, 1H) 5.28 (s, 1H)6.40 (t, J=2.49 Hz, 1H) 6.91-6.94 (m, 1H) 6.96-7.00 (m, 1H) 7.03 (d,J=1.94 Hz, 1H). MS (M+H⁺) 458.1.

Example ER-895088-01 was prepared from compound ER-895305 (25.0 mg,0.058 mmol) and commercially available (S)-tert-butyl3-methylpiperazine-1-carboxylate (35.2 mg, 0.177 mmol) in a mannersimilar to that of example ER-894463. Purification by LCMS, using HPLCcondition III, afforded the desired product (9.3 mg, 29%).

Example ER-895116 was prepared from compound ER-895305 (25.0 mg, 0.058mmol) and commercially available (R)-tert-butyl pyrrolidin-3-ylcarbamate(36.5 mg, 0.196 mmol) in a manner similar to that of example ER-894463.Purification by LCMS, using HPLC condition III, afforded the desiredproduct (16 mg, 52%).

Example ER-895089 was prepared from compound ER-895305 (25.0 mg, 0.058mmol) and commercially available (S)-tert-butyl pyrrolidin-3-ylcarbamate(36.7 mg, 0.197 mmol) in a manner similar to that of example ER-894463.Purification by LCMS, using HPLC condition III, afforded the desiredproduct (7.1 mg, 23%).

Example ER-895090 was prepared from compound ER-895305 (25.0 mg, 0.058mmol) and commercially available (1S,4S)-tert-butyl2,5-diazabicyclo[2.2.1]heptane-2-carboxylate (38.6 mg, 0.196 mmol) in amanner similar to that of example ER-894463. Purification by LCMS, usingHPLC condition III, afforded the desired product (18.1 mg, 57%).

Example ER-895115 was prepared from compound ER-895305 (25.6 mg, 0.060mmol) and commercially available (S)-tert-butyl3-aminopyrrolidine-1-carboxylate (31.5 mg, 0.169 mmol) in a mannersimilar to that of example ER-894463. Purification by LCMS, using HPLCcondition III, afforded the desired product (3.5 mg, 11%).

Example ER-895745 was prepared from compound ER-895305 (26.2 mg, 0.061mmol) and commercially available tert-butylhexahydropyrrolo[3,4-c]pyrrole-2(1H)-carboxylate (32.9 mg, 0.155 mmol)in a manner similar to that of example ER-894463. Purification by LCMS,using HPLC condition III, afforded the desired product (18.1 mg, 53%).

Example ER-895091-01 was prepared from compound4-(7-(difluoromethyl)-5-(3,4-dimethoxyphenyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidin-2-yl)-2-fluorobenzoicacid (25.0 mg, 0.056 mmol) and commercially available tert-butylpiperazine-1-carboxylate (21.0 mg, 0.113 mmol) in a manner similar tothat of example ER-894463. Purification by LCMS, using HPLC conditionIII, afforded the desired product (2.5 mg, 8%).

Example ER-895092 was prepared from compound4-(7-(difluoromethyl)-5-(3,4-dimethoxyphenyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidin-2-yl)-2-fluorobenzoicacid (25.0 mg, 0.056 mmol) and commercially available tert-butyl4-aminopiperidine-1-carboxylate (22.0 mg, 0.110 mmol) in a mannersimilar to that of example ER-894463. Purification by LCMS, using HPLCcondition III, afforded the desired product (5.2 mg, 16%).

Example ER-895093 was prepared from compound4-(7-(difluoromethyl)-5-(3,4-dimethoxyphenyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidin-2-ye-2-fluorobenzoicacid (25.0 mg, 0.056 mmol) and commercially available (S)-tert-butyl3-aminopiperidine-1-carboxylate (22.0 mg, 0.110 mmol) in a mannersimilar to that of example ER-894463. Purification by LCMS, using HPLCcondition III, afforded the desired product (10.3 mg, 32%).

Example ER-895094 was prepared from compound4-(7-(difluoromethyl)-5-(3,4-dimethoxyphenyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidin-2-yl)-2-fluorobenzoicacid (25.0 mg, 0.056 mmol) and commercially available (R)-tert-butyl3-aminopiperidine-1-carboxylate (22.0 mg, 0.110 mmol) in a mannersimilar to that of example ER-894463. Purification by LCMS, using HPLCcondition III, afforded the desired product (7.4 mg, 23%).

Example ER-895096 was prepared from compound4-(7-(difluoromethyl)-5-(3,4-dimethoxyphenyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidin-2-yl)-2-fluorobenzoicacid (25.0 mg, 0.056 mmol) and commercially available (2S,5R)-tert-butyl2,5-dimethylpiperazine-1-carboxylate (24.0 mg, 0.113 mmol) in a mannersimilar to that of example ER-894463. Purification by LCMS, using HPLCcondition III, afforded the desired product (13.9 mg, 43%).

Example ER-895097 was prepared from compound4-(7-(difluoromethyl)-5-(3,4-dimethoxyphenyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidin-2-yl)-2-fluorobenzoicacid (25.0 mg, 0.056 mmol) and commercially available (R)-tert-butyl3-aminopyrrolidine-1-carboxylate (21.0 mg, 0.113 mmol) in a mannersimilar to that of example ER-894463. Purification by LCMS, using HPLCcondition III, afforded the desired product (17.3 mg, 56%).

Example ER-895098 was prepared from compound4-(7-(difluoromethyl)-5-(3,4-dimethoxyphenyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidin-2-yl)-2-fluorobenzoicacid (25.0 mg, 0.056 mmol) and commercially available (S)-tert-butyl3-aminopyrrolidine-1-carboxylate (21.0 mg, 0.113 mmol) in a mannersimilar to that of example ER-894463. Purification by LCMS, using HPLCcondition III, afforded the desired product (18.5 mg, 60%).

Example ER-895101 was prepared from compound4-(7-(difluoromethyl)-5-(3,4-dimethoxyphenyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidin-2-yl)-2-fluorobenzoicacid (25.0 mg, 0.056 mmol) and commercially available (1S,4S)-tert-butyl2,5-diazabicyclo[2.2.1]heptane-2-carboxylate (22.0 mg, 0.112 mmol) in amanner similar to that of example ER-894463. Purification by LCMS, usingHPLC condition III, afforded the desired product (9 mg, 28%).

Example ER-895099 was prepared from compound4-(7-(difluoromethyl)-5-(3,4-dimethoxyphenyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidin-2-yl)-2-fluorobenzoicacid (25.0 mg, 0.056 mmol) and commercially available tert-butyl3-aminoazetidine-1-carboxylate (19.0 mg, 0.110 mmol) in a manner similarto that of example ER-895080. Purification by LCMS, using HPLC conditionIII, afforded the desired product (14.1 mg, 41%).

Example ER-895102 was prepared from compound4-(7-(difluoromethyl)-5-(3,4-dimethoxyphenyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidin-2-yl)-3-fluorobenzoicacid (25.0 mg, 0.056 mmol) and commercially available tert-butylpiperazine-1-carboxylate (21.0 mg, 0.113 mmol) in a manner similar tothat of example ER-894463. Purification by LCMS, using HPLC conditionIII, afforded the desired product (17.2 mg, 56%).

Example ER-895104 was prepared from compound4-(7-(difluoromethyl)-5-(3,4-dimethoxyphenyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidin-2-yl)-3-fluorobenzoicacid (25.0 mg, 0.056 mmol) and commercially available (S)-tert-butyl3-aminopiperidine-1-carboxylate (22.0 mg, 0.110 mmol) in a mannersimilar to that of example ER-894463. Purification by LCMS, using HPLCcondition III, afforded the desired product (17.1 mg, 54%).

Example ER-89510 was prepared from compound4-(7-(difluoromethyl)-5-(3,4-dimethoxyphenyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidin-2-yl)-3-fluorobenzoicacid (25.0 mg, 0.056 mmol) and commercially available (R)-tert-butyl3-aminopiperidine-1-carboxylate (22.0 mg, 0.110 mmol) in a mannersimilar to that of example ER-894463. Purification by LCMS, using HPLCcondition III, afforded the desired product (15.8 mg, 50%).

Example ER-895106 was prepared from compound4-(7-(difluoromethyl)-5-(3,4-dimethoxyphenyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidin-2-yl)-3-fluorobenzoicacid (25.0 mg, 0.056 mmol) and commercially available (S)-tert-butyl3-methylpiperazine-1-carboxylate (22.0 mg, 0.110 mmol) in a mannersimilar to that of example ER-894463. Purification by LCMS, using HPLCcondition III, afforded the desired product (14.9 mg, 47%).

Example ER-895107 was prepared from compound4-(7-(difluoromethyl)-5-(3,4-dimethoxyphenyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidin-2-ye-3-fluorobenzoicacid (25.0 mg, 0.056 mmol) and commercially available (2S,5R)-tert-butyl2,5-dimethylpiperazine-1-carboxylate (24.0 mg, 0.112 mmol) in a mannersimilar to that of example ER-894463. Purification by LCMS, using HPLCcondition III, afforded the desired product (14.2 mg, 44%).

Example ER-895109 was prepared from compound4-(7-(difluoromethyl)-5-(3,4-dimethoxyphenyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidin-2-yl)-3-fluorobenzoicacid (25.0 mg, 0.056 mmol) and commercially available (S)-tert-butyl3-aminopyrrolidine-1-carboxylate (21.0 mg, 0.113 mmol) in a mannersimilar to that of example ER-894463. Purification by LCMS, using HPLCcondition III, afforded the desired product (17.7 mg, 57%).

Example ER-895112 was prepared from compound4-(7-(difluoromethyl)-5-(3,4-dimethoxyphenyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidin-2-yl)-3-fluorobenzoicacid (25.0 mg, 0.056 mmol) and commercially available (1S,4S)-tert-butyl2,5-diazabicyclo[2.2.1]heptane-2-carboxylate (22.0 mg, 0.111 mmol) in amanner similar to that of example ER-894463. Purification by LCMS, usingHPLC condition III, afforded the desired product (14.4 mg, 46%).

Example ER-895111 was prepared from compound4-(7-(difluoromethyl)-5-(3,4-dimethoxyphenyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidin-2-yl)-3-fluorobenzoicacid (25.0 mg, 0.056 mmol) and commercially available tert-butyl2,6-diazaspiro[3.3]heptane-2-carboxylate (22.0 mg, 0.111 mmol) in amanner similar to that of example ER-895080. Purification by LCMS, usingHPLC condition III, afforded the desired product (22.7 mg, 63%).

Example ER-895731 was prepared from compound3-(7-(difluoromethyl)-5-(3,4-dimethoxyphenyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidin-2-yl)benzoicacid (25.0 mg, 0.058 mmol) and commercially available tert-butylpiperazine-1-carboxylate (32.8 mg, 0.176 mmol) in a manner similar tothat of example ER-894463. Purification by LCMS, using HPLC conditionIII, afforded the desired product (22.1 mg, 70%).

Example ER-895732 was prepared from compound3-(7-(difluoromethyl)-5-(3,4-dimethoxyphenyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidin-2-yl)benzoicacid (25.0 mg, 0.058 mmol) and commercially available tert-butyl4-aminopiperidine-1-carboxylate (31.0 mg, 0.155 mmol) in a mannersimilar to that of example ER-894463. Purification by LCMS, using HPLCcondition III, afforded the desired product (22 mg, 70%).

Example ER-895733 was prepared from compound3-(7-(difluoromethyl)-5-(3,4-dimethoxyphenyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidin-2-yl)benzoicacid (25.0 mg, 0.058 mmol) and commercially available (S)-tert-butyl3-aminopiperidine-1-carboxylate (34.0 mg, 0.170 mmol) in a mannersimilar to that of example ER-894463. Purification by LCMS, using HPLCcondition III, afforded the desired product (26.5 mg, 85%).

Example ER-895734 was prepared from compound3-(7-(difluoromethyl)-5-(3,4-dimethoxyphenyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidin-2-yl)benzoicacid (25.0 mg, 0.058 mmol) and commercially available (R)-tert-butyl3-aminopiperidine-1-carboxylate (36.3 mg, 0.181 mmol) in a mannersimilar to that of example ER-894463. Purification by LCMS, using HPLCcondition III, afforded the desired product (23.4 mg, 75%).

Example ER-895739 was prepared from compound3-(7-(difluoromethyl)-5-(3,4-dimethoxyphenyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidin-2-yl)benzoicacid (25.0 mg, 0.058 mmol) and commercially available (S)-tert-butyl3-aminopyrrolidine-1-carboxylate (37.7 mg, 0.202 mmol) in a mannersimilar to that of example ER-894463. Purification by LCMS, using HPLCcondition III, afforded the desired product (22.9 mg, 73%).

Example ER-895740 was prepared from compound3-(7-(difluoromethyl)-5-(3,4-dimethoxyphenyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidin-2-yl)benzoicacid (25.0 mg, 0.058 mmol) and commercially available (R)-tert-butyl3-aminopyrrolidine-1-carboxylate (34.8 mg, 0.187 mmol) in a mannersimilar to that of example ER-894463. Purification by LCMS, using HPLCcondition III, afforded the desired product (23.1 mg, 73%).

Example ER-895744 was prepared from compound3-(7-(difluoromethyl)-5-(3,4-dimethoxyphenyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidin-2-yl)benzoicacid (25.0 mg, 0.058 mmol) and commercially available tert-butylhexahydropyrrolo[3,4-c]pyrrole-2(1H)-carboxylate (26.9 mg, 0.127 mmol)in a manner similar to that of example ER-894463. Purification by LCMS,using HPLC condition III, afforded the desired product (26.5 mg, 82%).

Example ER-895741 was prepared from compound3-(7-(difluoromethyl)-5-(3,4-dimethoxyphenyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidin-2-yl)benzoicacid (25.0 mg, 0.058 mmol) and commercially available tert-butyl3-aminoazetidine-1-carboxylate (30.1 mg, 0.175 mmol) in a manner similarto that of example ER-895080. Purification by LCMS, using HPLC conditionIII, afforded the desired product (5.5 mg, 16%).

Example ER-895718 was prepared from compound4-(7-(difluoromethyl)-5-(3,4-dimethoxyphenyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidin-2-yl)-3-methylbenzoicacid (25.0 mg, 0.056 mmol) and commercially available tert-butyl4-aminopiperidine-1-carboxylate (45.4 mg, 0.227 mmol) in a mannersimilar to that of example ER-894463. Purification by LCMS, using HPLCcondition III, afforded the desired product (21.4 mg, 68%).

Example ER-895719 was prepared from compound4-(7-(difluoromethyl)-5-(3,4-dimethoxyphenyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidin-2-yl)-3-methylbenzoicacid (25.0 mg, 0.056 mmol) and commercially available (S)-tert-butyl3-aminopiperidine-1-carboxylate (29.8 mg, 0.149 mmol) in a mannersimilar to that of example ER-894463. Purification by LCMS, using HPLCcondition III, afforded the desired product (21.6 mg, 69%).

Example ER-895720 was prepared from compound4-(7-(difluoromethyl)-5-(3,4-dimethoxyphenyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidin-2-yl)-3-methylbenzoicacid (25.0 mg, 0.056 mmol) and commercially available (R)-tert-butyl3-aminopiperidine-1-carboxylate (28.6 mg, 0.143 mmol) in a mannersimilar to that of example ER-894463. Purification by LCMS, using HPLCcondition III, afforded the desired product (21.7 mg, 69%).

Example ER-895721 was prepared from compound4-(7-(difluoromethyl)-5-(3,4-dimethoxyphenyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidin-2-yl)-3-methylbenzoicacid (25.0 mg, 0.056 mmol) and commercially available (S)-tert-butyl3-methylpiperazine-1-carboxylate (31.1 mg, 0.155 mmol) in a mannersimilar to that of example ER-894463. Purification by LCMS, using HPLCcondition III, afforded the desired product (20.2 mg, 64%).

Example ER-895722 was prepared from compound4-(7-(difluoromethyl)-5-(3,4-dimethoxyphenyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidin-2-yl)-3-methylbenzoicacid (25.0 mg, 0.056 mmol) and commercially available (R)-tert-butyl3-methylpiperazine-1-carboxylate (41.2 mg, 0.206 mmol) in a mannersimilar to that of example ER-894463. Purification by LCMS, using HPLCcondition III, afforded the desired product (21 mg, 67%).

Example ER-895723 was prepared from compound4-(7-(difluoromethyl)-5-(3,4-dimethoxyphenyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidin-2-yl)-3-methylbenzoicacid (25.0 mg, 0.056 mmol) and commercially available (2S,5R)-tert-butyl2,5-dimethylpiperazine-1-carboxylate (34.1 mg, 0.159 mmol) in a mannersimilar to that of example ER-894463. Purification by LCMS, using HPLCcondition III, afforded the desired product (20.5 mg, 64%).

Example ER-895725 was prepared from compound4-(7-(difluoromethyl)-5-(3,4-dimethoxyphenyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidin-2-yl)-3-methylbenzoicacid (25.0 mg, 0.056 mmol) and commercially available (S)-tert-butyl3-aminopyrrolidine-1-carboxylate (43.7 mg, 0.235 mmol) in a mannersimilar to that of example ER-894463. Purification by LCMS, using HPLCcondition III, afforded the desired product (19.8 mg, 65%).

Example ER-895726—was prepared from compound4-(7-(difluoromethyl)-5-(3,4-dimethoxyphenyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidin-2-yl)-3-methylbenzoicacid (25.0 mg, 0.056 mmol) and commercially available (R)-tert-butyl3-aminopyrrolidine-1-carboxylate (31.0 mg, 0.167 mmol) in a mannersimilar to that of example ER-894463. Purification by LCMS, using HPLCcondition III, afforded the desired product (20.6 mg, 67%).

Example ER-895729 was prepared from compound4-(7-(difluoromethyl)-5-(3,4-dimethoxyphenyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidin-2-yl)-3-methylbenzoicacid (25.0 mg, 0.056 mmol) and commercially available (1S,4S)-tert-butyl2,5-diazabicyclo[2.2.1]heptane-2-carboxylate (29.9 mg, 0.151 mmol) in amanner similar to that of example ER-894463. Purification by LCMS, usingHPLC condition III, afforded the desired product (19.6 mg, 62%).

Example ER-895730 was prepared from compound4-(7-(difluoromethyl)-5-(3,4-dimethoxyphenyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidin-2-yl)-3-methylbenzoicacid (25.0 mg, 0.056 mmol) and commercially available tert-butylhexahydropyrrolo[3,4-c]pyrrole-2(1H)-carboxylate (28.9 mg, 0.136 mmol)in a manner similar to that of example ER-894463. Purification by LCMS,using HPLC condition III, afforded the desired product (23.9 mg, 74%).

Example ER-895755 was prepared from compound4-(7-(difluoromethyl)-5-(3,4-dimethoxyphenyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidin-2-yl)-3-methylbenzoicacid (25.5 mg, 0.058 mmol) and commercially available tert-butylpiperazine-1-carboxylate (27.9 mg, 0.150 mmol) in a manner similar tothat of example ER-894463. Purification by LCMS, using HPLC conditionIII, afforded the desired product (28.3 mg, 89%).

Example ER-895727 was prepared from compound4-(7-(difluoromethyl)-5-(3,4-dimethoxyphenyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidin-2-yl)-3-methylbenzoicacid (25.0 mg, 0.056 mmol) and commercially available tert-butyl3-aminoazetidine-1-carboxylate (37.0 mg, 0.215 mmol) in a manner similarto that of example ER-895080. Purification by LCMS, using HPLC conditionIII, afforded the desired product (22.3 mg, 65%).

Example ER-896059 was prepared from compound ER-895435 (10.0 mg, 0.023mmol) and commercially available tert-butyl4-aminopiperidine-1-carboxylate (17.8 mg, 0.089 mmol) in a mannersimilar to that of example ER-894463. Purification by LCMS, using HPLCcondition III, afforded the desired product (11.2 mg, 87%).

Example ER-896060 was prepared from compound ER-895435 (25.0 mg, 0.056mmol) and commercially available (S)-tert-butyl3-aminopiperidine-1-carboxylate (19.3 mg, 0.096 mmol) in a mannersimilar to that of example ER-894463. Purification by LCMS, using HPLCcondition III, afforded the desired product (12.3 mg, 95%).

Example ER-896061 was prepared from compound ER-895435 (25.0 mg, 0.056mmol) and commercially available (R)-tert-butyl3-aminopiperidine-1-carboxylate (16.9 mg, 0.084 mmol) in a mannersimilar to that of example ER-894463. Purification by LCMS, using HPLCcondition III, afforded the desired product (12.2 mg, 94%).

Example ER-896062 was prepared from compound ER-895435 (25.0 mg, 0.056mmol) and commercially available (S)-tert-butyl3-methylpiperazine-1-carboxylate (23.9 mg, 0.119 mmol) in a mannersimilar to that of example ER-894463. Purification by LCMS, using HPLCcondition III, afforded the desired product (10.7 mg, 83%).

Example ER-896063 was prepared from compound ER-895435 (25.0 mg, 0.056mmol) and commercially available (R)-tert-butyl3-methylpiperazine-1-carboxylate (20.5 mg, 0.102 mmol) in a mannersimilar to that of example ER-894463. Purification by LCMS, using HPLCcondition III, afforded the desired product (10.9 mg, 84%).

Example ER-896064 was prepared from compound ER-895435 (25.0 mg, 0.056mmol) and commercially available (2S,5R)-tert-butyl2,5-dimethylpiperazine-1-carboxylate (17.2 mg, 0.080 mmol) in a mannersimilar to that of example ER-894463. Purification by LCMS, using HPLCcondition III, afforded the desired product (10.3 mg, 78%).

Example ER-896067 was prepared from compound ER-895435 (25.0 mg, 0.056mmol) and commercially available (1S,4S)-tert-butyl2,5-diazabicyclo[2.2.1]heptane-2-carboxylate (15.2 mg, 0.077 mmol) in amanner similar to that of example ER-894463. Purification by LCMS, usingHPLC condition III, afforded the desired product (9.9 mg, 77%).

Example ER-896068 was prepared from compound ER-895435 (25.0 mg, 0.056mmol) and commercially available tert-butylhexahydropynolo[3,4-c]pyrrole-2(1H)-carboxylate (17.3 mg, 0.081 mmol) ina manner similar to that of example ER-894463. Purification by LCMS,using HPLC condition III, afforded the desired product (12.3 mg, 93%).

Example ER-896071 was prepared from compound ER-895435 (25.9 mg, 0.060mmol) and commercially available tert-butyl3-(aminomethyl)azetidine-1-carboxylate (30.4 mg, 0.163 mmol) in a mannersimilar to that of example ER-895080. Purification by LCMS, using HPLCcondition III, afforded the desired product (18.6 mg, 51%).

Synthetic Examples Section G Preparation of ER-893993

A 500 mL flask was charged with4,4,4′,4′,5,5,5′,5′-octamethyl-2,2′-bi(1,3,2-dioxaborolane) (10.78 g, 42mmol),5-(3,4-dimethoxyphenyl)-7-(trifluoromethyl)pyrazolo[1,5-a]pyrimidin-2-yltrifluoromethanesulfonate (10.0 g, 21 mmol), Potassium acetate (10.41 g,106 mmol), and 1,4-dioxane (100 ml). The reaction mixture was degassedwith nitrogen for 15 minutes.1,1′-Bis(diphenylphosphino)ferrocene-palladium(II) dichloridedichloromethane complex (4.33 g, 5.3 mmol) was added and the mixture wassealed and stirred for 3 hours at 80° C. The reaction mixture was cooledat room temperature and was partitioned between EtOAc (1000 mL) and sat.NaHCO3 solution (200 mL). Phases were separated and the aqueous was backextract 2× with EtOAc (200 mL). The combined organic layers were washedwith brine (200 mL). The organic layer was dried with anhydrous sodiumsulfate, filtered and concentrated. Crude material was dissolved inEtOAc (500 mL), Heptane (500 mL) was added and the black solid impuritywas removed by filtration. The filtrate containing the desired productwas concentrated under reduced pressure, was suspended in 150 mL of IPA,was heated at 70° C. for 30 minutes and then cooled at room temperature.The green precipitate was filtered off, rinsed with IPA and dried onvacuum pump.5-(3,4-dimethoxyphenyl)-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-7-(trifluoromethyl)pyrazolo[1,5-a]pyrimidine(4.50 g, 10.02 mmol, 47.2% yield)

In a 25 mL flask and under an atmosphere of nitrogen, to a solution oftert-butyl 2-methyl-4-oxopiperidine-1-carboxylate (558 mg, 2.6 mmol) and1,1,1-Trifluoro-N-phenyl-N-(trifluoromethyl)sulfonyl methanesulfonamide(1869 mg, 5.2 mmol) in anhydrous THF (5 mL) at −78° C. was added 1.0MLiHMDS in THF (5.23 mL, 5.2 mmol). The mixture was stirred overnightwhile slowly being allowed to warm at room temperature. The reaction wasquenched with saturated NaHCO3. Then the mixture was extracted 3× withMTBE. The combined organic layers were dried with sodium sulfate,filtered and concentrated. A mixture of regioisomers (tert-butyl6-methyl-4-(((trifluoromethyl)sulfonyl)oxy)-5,6-dihydropyridine-1(2H)-carboxylateand tert-butyl2-methyl-4-(((trifluoromethyl)sulfonyl)oxy)-5,6-dihydropyridine-1(2H)-carboxylate)was obtained as a pale yellow oil (397 mg, 44% yield).

A 5-10 mL microwave vial was charged with5-(3,4-dimethoxyphenyl)-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-7-(trifluoromethyl)pyrazolo[1,5-a]pyrimidine(650 mg, 1.45 mmol), tert-butyl2-methyl-4-(((trifluoromethyl)sulfonyl)oxy)-5,6-dihydropyridine-1(2H)-carboxylate(mixture with tert-butyl6-methyl-4-(((trifluoromethyl)sulfonyl)oxy)-5,6-dihydropyridine-1(2H)-carboxylate,total is 396 mg, 1.15 mmol), Potassium carbonate (792 mg, 5.7 mmol),Water (2.0 ml) and THF (4.5 ml). The reaction mixture was degassed withnitrogen for 15 minutes.1,1′-Bis(diphenylphosphino)ferrocene-palladium(II) dichloridedichloromethane complex (234 mg, 0.29 mmol) was added and the mixturewas sealed and stirred overnight at 90° C. The reaction mixture wascooled at room temperature and concentrated. The crude product waspurified on silica-gel (Column Interchim 40 g, 30 μM) eluting with12-100% EtOAc/Heptane. A mixture of regioisomers (tert-butyl4-(5-(3,4-dimethoxyphenyl)-7-(trifluoromethyl)pyrazolo[1,5-a]pyrimidin-2-yl)-2-methyl-5,6-dihydropyridine-1(2H)-carboxylate and tert-butyl4-(5-(3,4-dimethoxyphenyl)-7-(trifluoromethyl)pyrazolo[1,5-a]pyrimidin-2-yl)-6-methyl-5,6-dihydropyridine-1(2H)-carboxylate)was obtained as a yellow solid (142 mg, 24% yield).

Synthesis of ER-896993

Sodium tetrahydroborate (10.94 mg, 0.29 mmol) was slowly added to asuspension of tert-butyl4-(5-(3,4-dimethoxyphenyl)-7-(trifluoromethyl)pyrazolo[1,5-a]pyrimidin-2-yl)-2-methyl-5,6-dihydropyridine-1(2H)-carboxylate(Mixture with regioisomer tert-butyl4-(5-(3,4-dimethoxyphenyl)-7-(trifluoromethyl)pyrazolo[1,5-a]pyrimidin-2-yl)-6-methyl-5,6-dihydropyridine-1(2H)-carboxylate,total is 50 mg, 0.096 mmol) in Ethanol (1.0 ml) at room temperature. Themixture was heated to 80° C. for 60 minutes. Ethyl acetate was added.The mixture was sequentially washed with an aqueous saturated solutionof NH₄Cl and then brine.

The organic layer was dried with anhydrous sodium sulfate, was filteredand was concentrated. The crude product was purified on silica-gel(Column Interchim 25 g, 30 μM) eluting with 12-100% EtOAc/Heptane. Amixture of regioisomers (tert-butyl4-(5-(3,4-dimethoxyphenyl)-7-(trifluoromethyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidin-2-yl)-2-methyl-5,6-dihydropyridine-1(2H)-carboxylateand tert-butyl4-(5-(3,4-dimethoxyphenyl)-7-(trifluoromethyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidin-2-yl)-6-methyl-5,6-dihydropyridine-1(2H)-carboxylate)was obtained as a white solid (38 mg, 75% yield).

Tert-butyl4-(5-(3,4-dimethoxyphenyl)-7-(trifluoromethyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidin-2-yl)-2-methyl-5,6-dihydropyridine-1(2H)-carboxylate (Mixture with regioisomer tert-butyl4-(5-(3,4-dimethoxyphenyl)-7-(trifluoromethyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidin-2-yl)-6-methyl-5,6-dihydropyridine-1(2H)-carboxylate,total is 34 mg, 0.065 mmol) was dissolved in Ethanol (0.5 ml) and 4.00MHCl in Dioxane (0.5 ml) and the mixture was stirred at 40° C. for 1hour. 1 mL of toluene was added. The reaction mixture was concentrated.

The product was dried on vacuum pump to afford 29 mg of the finalproduct as HCl salt (mixture of regioisomers5-(3,4-dimethoxyphenyl)-2-(6-methyl-1,2,3,6-tetrahydropyridin-4-yl)-7-(trifluoromethyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidinehydrochloride and5-(3,4-dimethoxyphenyl)-2-(2-methyl-1,2,3,6-tetrahydropyridin-4-yl)-7-(trifluoromethyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidinehydrochloride) (ER-896993).

Synthesis of ER-896994

Tert-butyl4-(5-(3,4-dimethoxyphenyl)-7-(trifluoromethyl)pyrazolo[1,5-a]pyrimidin-2-yl)-2-methyl-5,6-dihydropyridine-1(2H)— carboxylate (Mixture with regioisomer tert-butyl4-(5-(3,4-dimethoxyphenyl)-7-(trifluoromethyl)pyrazolo[1,5-a]pyrimidin-2-yl)-6-methyl-5,6-dihydropyridine-1(2H)-carboxylate,total is 60 mg, 0.116 mmol) was dissolved in Methanol (2.5 ml). Thesolution was treated with Hydrogen for 2.5 hrs at room temperature usingH-Cube (Full H2, 1 mL/min, small size (30 mm) 10% Palladium on CarbonCatCart). 1 mL Toluene was added. Solvent was concentrated. The crudeproduct was purified on silica-gel (Column Interchim 25 g, 30 μM)eluting with 12-100% EtOAc/Heptane to afford tert-butyl4-(5-(3,4-dimethoxyphenyl)-7-(trifluoromethyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidin-2-yl)-2-methylpiperidine-1-carboxylate(36 mg, 0.069 mmol, 59% yield) as a white solid.

Tert-butyl4-(5-(3,4-dimethoxyphenyl)-7-(trifluoromethyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidin-2-yl)-2-methylpiperidine-1-carboxylate(29 mg, 0.055 mmol) was dissolved in Ethanol (0.5 ml) and 4.00M HCl inDioxane (0.5 ml) and the mixture was stifled at 40° C. for 1 hour. 1 mLof toluene was added. The reaction mixture was concentrated. The productwas dried on vacuum pump to afford 25 mg of the final product as HClsalt(5-(3,4-dimethoxyphenyl)-2-(2-methylpiperidin-4-yl)-7-(trifluoromethyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidinehydrochloride, ER-896994).

Example ER-897090

Example ER-897090 was prepared from5-(3,4-dimethoxyphenyl)-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-7-(trifluoromethyl)pyrazolo[1,5-a]pyrimidine(507 mg, 1.129 mmol) and tert-butyl4-(((trifluoromethyl)sulfonyl)oxy)-2,3,6,7-tetrahydro-1H-azepine-1-carboxylate(300 mg, 0.869 mmol) prepared from commercially available tert-butyl4-oxoazepane-1-carboxylate in a manner similar to that of ER-896993(Section G) except the deprotection step was carried out in a mannersimilar to that of ER-887084 (Section B) to afford ER-897090 (96 mg, 26%yield). LC-MS: Rt 1.49 min, (M+1)⁺423.06 under condition II.

Compound ER-897090 (94 mg, 0.224 mmol) was resolved into its constituentenantiomers in a manner similar to that of ER-890044 (Section A) toafford one of the isomer ER-897212. (17 mg, 18% yield).

Example ER-897130 was prepared from5-(3,4-dimethoxyphenyl)-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-7-(trifluoromethyl)pyrazolo[1,5-a]pyrimidine(493 mg, 1.097 mmol) and tert-butyl3-(((trifluoromethyl)sulfonyl)oxy)-2,5-dihydro-1H-pyrrole-1-carboxylate(174 mg, 0.548 mmol) prepared from commercially available tert-butyl3-oxopyrrolidine-1-carboxylate in a manner similar to that of ER-896993(Section G) except the deprotection step was carried out in a mannersimilar to that of ER-887084 (Section B) to afford ER-897130 (68 mg, 31%yield).

Example ER-897142 was prepared from5-(3,4-dimethoxyphenyl)-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-7-(trifluoromethyl)pyrazolo[1,5-a]pyrimidine(489 mg, 1.089 mmol) and4-((tert-butoxycarbonyl)amino)cyclohex-1-en-1-yltrifluoromethanesulfonate (188 mg, 0.544 mmol) prepared fromcommercially available tert-butyl(4-oxocyclohexyl)carbamate in a mannersimilar to that of ER-896993 (Section G) except the deprotection stepwas carried out in a manner similar to that of ER-887084 (Section B) toafford ER-897142 (111 mg, 48% yield). (LC-MS: Rt 1.57 min, (M+1)⁺423.06under condition II)

Example ER-897364 and ER-897365

Compound ER-897090 (100 mg, 0.237 mmol) was resolved into itsconstituent enantiomers in a manner similar to that of ER-890044(Section A) to afford ER-897364 (4 mg, 4% yield, >95% ee), and ER-897365(6.4 mg, 6% yield, >95% ee).

Example ER-897547 Preparation of tert-butyl3-methyl-4-oxopiperidine-1-carboxylate

A solution of 1-benzyl-3-methylpiperidin-4-one (1.63 g, 8.018 mmol) anddi-tert-butyl dicarbonate (1.925 g, 8.82 mmol) in 50 ml of Methanol washydrogenated using H-Cube (Controlled H2 at 50 bar, flow rate 1.0ml/min) with Pd—C cartridge. The reaction mixture was recirculated for12 hours. After washing the H-Cube thoroughly with Methanol, the solventwas evaporated and the resulting oil was purified by silica gelchromatography to afford the title compound (1.5 g, 88%).

Example ER-897547 was prepared from5-(3,4-dimethoxyphenyl)-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-7-(trifluoromethyl)pyrazolo[1,5-a]pyrimidine(488 mg, 1.086 mmol) and tert-butyl5-methyl-4-(((trifluoromethyl)sulfonyl)oxy)-5,6-dihydropyridine-1(2H)-carboxylate(250 mg, 0.724 mmol) prepared from tert-butyl3-methyl-4-oxopiperidine-1-carboxylate in a manner similar to that ofER-896993 (Section G) except the deprotection step was carried out in amanner similar to that of ER-887084 (Section B) to afford ER-897547 (68mg, 31% yield).

Example ER-897597 was prepared from5-(3,4-dimethoxyphenyl)-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-7-(trifluoromethyl)pyrazolo[1,5-a]pyrimidine(475 mg, 1.058 mmol) and commercially available tert-butyl4-(4-bromophenyl)piperidine-1-carboxylate (240 mg, 0.705 mmol) in amanner similar to that of ER-896993 (Section G) except the deprotectionstep was carried out in a manner similar to that of ER-887084 (SectionB) to afford ER-897597 (32 mg, 9% yield). LC-MS: Rt 1.72 min,(M+1)⁺486.95 under condition II.

Example ER-897814 and ER-897815

Compound ER-897597 (20 mg, 0.041 mmol) was resolved into its constituentenantiomers in a manner similar to that of ER-890044 (Section A) toafford ER-897814 (7 mg, 35% yield, >95% ee), and ER-897815 (3 mg, 15%yield, >95% ee).

Example ER-897728 Preparation of benzyl9-oxo-3-azaspiro[5.5]undec-7-ene-3-carboxylate

Benzyl 4-formylpiperidine-1-carboxylate (5.17 g, 20.907 mmol) andp-Toluenesulfonic acid monohydrate (0.398 g, 2.091 mmol) were stirred inBenzene (30.0 ml, 334.136 mmol) at 70° C. But-3-en-2-one (3.76 ml,41.813 mmol) was added and the reaction mixture was refluxed o/n whileremoving water with dean-stark trap. After cooling the reaction mixtureto rt, sat. NaHCO3 soln. was added and the organic layer was dried overNa2SO4 and evaporated. The resulting oil was purified by Biotage (SiO2,250 g, EtOAc/Hep 10% to 50%) to afford the title compound (3.58 g, 11.96mmol, 57.2% yield).

Preparation of tert-butyl 9-oxo-3-azaspiro[5.5]undecane-3-carboxylate

A solution of benzyl 9-oxo-3-azaspiro[5.5]undec-7-ene-3-carboxylate(1.26 g, 4.209 mmol), di-tert-butyl dicarbonate (1.01 g, 4.63 mmol) andTEA (1.47 ml, 10.522 mmol) in 50 ml of Methanol was hydrogenated usingH-Cube (Full H2, flow rate 1.0 ml/min) with Pd—C cartridge. The reactionmixture was recirculated for 5 hours. After washing the H-Cubethoroughly with Methanol, the solvent was evaporated and the resultingoil was purified by silica gel chromatography to afford the titlecompound (681 mg, 61%).

Example ER-897728 was prepared from5-(3,4-dimethoxyphenyl)-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-7-(trifluoromethyl)pyrazolo[1,5-a]pyrimidine(489 mg, 1.089 mmol) and tert-butyl9-(((trifluoromethyl)sulfonyl)oxy)-3-azaspiro[5.5]undec-8-ene-3-carboxylate(290 mg, 0.726 mmol) prepared from tert-butyl9-oxo-3-azaspiro[5.5]undecane-3-carboxylate in a manner similar to thatof ER-896993 (Section G) except the deprotection step was carried out ina manner similar to that of ER-887084 (Section B) to afford ER-897728(91 mg, 26% yield). LC-MS: Rt 1.67 min, (M+1)⁺477.13 under condition II.

Example ER-897851

Compound ER-897728 (85 mg, 0.178 mmol) was resolved into its constituentenantiomers in a manner similar to that of ER-890044 (Section A) toafford one of the isomer ER-897851. (28 mg, 33% yield).

Example ER-890978 was prepared from compound G2 (600 mg, 1.34 mmol) andcommercially available 4-bromo-2-chloropyridine in a manner similar tothat of example ER-896993 to afford2-(2-chloropyridin-4-yl)-5-(3,4-dimethoxyphenyl)-7-(trifluoromethyl)pyrazolo[1,5-a]pyrimidine(311 mg, 54% yield), followed by NaBH₄ reduction (carried out on 300 mgof this material). Purification by silica gel chromatography affordedthe desired product (277 mg, 92% yield).

Synthetic Examples Section H Preparation 1:1-methyl-2-(propan-2-ylidene)hydrazine

Acetone (100 mL) was treated dropwise (syringe pump) over 45 minuteswith N-Methylhydrazine (20 mL), and the reaction solution was stirred atroom temperature for 45 minutes. The reaction solution was then heatedat 55° C. for an additional 15 minutes, cooled to room temperature,dried over sodium sulfate, filtered and concentrated via rotavap at 40°C. or less. The crude product was then purified by distillation at110-122° C. to afford the title compound as a colorless oil (14.2 g,43.9%).

¹HNMR (400 MHz, DMSO-d6) δ: 1.58 (s, 3H), 1.72, (s, 3H), 2.63, (s, 3H),5.21, (br s, 1H).

Preparation 2: 3-(ethoxymethylene)pentane-2,4-dione

A solution of Acetylacetone (40 mL), Ethyl orthoformate (95.4 mL) andAcetic anhydride (54.2 mL) was heated to reflux (150° C.) for 1 hour.The reaction solution was cooled to room temperature and was directlysubjected to flash column chromatography (E. Merck Silica Gel (˜120 g);Eluent: 100% EtOAc followed by 5% EtOH in EtOAc). Concentration of thefractions containing the pure product using a rotavap at 40° C. affordedthe title compound as a dark red viscous oil (10.4 g, 17.0%).

¹HNMR (400 MHz, CDCl₃) δ: 1.36, (t, 3H), 2.28, (s, 3H), 2.35, (s, 3H),4.22, (q, 2H), 7.67, s, 1H).

Preparation 3:3-((1-methyl-2-(propan-2-ylidene)hydrazinyl)methylene)pentane-2,4-dione

3-(ethoxymethylene)pentane-2,4-dione (Preparation 2, 8.6 g) wasdissolved in Ether (30 mL) and cooled to 0° C. The reaction solution wasthen slowly treated with 1-methyl-2-(propan-2-ylidene)hydrazine(Preparation 1, 4.8 g) dropwise over 5 minutes, and then warmed andstirred at room temperature overnight. The reaction solution waspurified directly by flash column chromatography (Biotage Quad 25;Eluent: 20% EtOH in EtOAc) to afford the title compound as a light redsolid. (9.1 g, 84.0%).

¹H NMR (400 MHz, DMSO-d6) δ: 1.82, (s, 3H), 1.89, (s, 3H), 2.05, (s,6H), 3.07, (s, 3H), 7.40, (s, 1H).

Preparation 4: 1-(1,3-dimethyl-1H-pyrazol-4-yl)ethanone

3-((1-methyl-2-(propan-2-ylidene)hydrazinyl)methylene)pentane-2,4-dione(Preparation 3, 9.1 g) was dissolved in Ethanol (20 mL) and treated with1M Hydrogen chloride in Water (20 mL), the reaction solution was stirredat room temperature for 15 minutes. The reaction solution was thenconcentrated under vacuum with a rotavap to approximately 20 mL totalvolume. The reaction solution was then treated with saturated aqueoussodium bicarbonate (45 mL), and extracted with methylene chloride (4×50mL) The combined organic extracts were dried over sodium sulfate,filtered and concentrated in vacuo afford the title compound as a lightorange solid. (6.2 g, 97%).

¹H NMR (400 MHz, CDCl₃) δ: 2.37, (s, 3H), 2.45, (s, 3H), 3.84, (s, 3H),7.75, (s, 1H)

Preparation 5:1-(1,3-dimethyl-1H-pyrazol-4-yl)-4,4,4-trifluoro-3-hydroxybut-2-en-1-one

1-(1,3-dimethyl-1H-pyrazol-4-yl)ethanone (Preparation 4, 4.46 g) wasdissolved in Methanol (21 mL) and 25% Sodium methoxide in Methanol(11.07 mL) was added. The mixture was stirred for 5 minutes, and Aceticacid, trifluoro-, ethyl ester (7.701 mL) was added. The mixture washeated to reflux at 75° C. and was stirred for 19 hours. The reactionsolution was purified directly by flash column chromatography (BiotageQuad 25; Eluent: 100% EtOAc followed by 5% EtOH in EtOAc and then 20%EtOH in EtOAc) affording the title compound (tautomer) as a light redfoamy solid (7.0 g, 92.3%).

¹H NMR (400 MHz, CDCl₃) δ: 2.33 (s, 3H), 3.76, (s, 3H), 6.10, (s, 1H),7.84, (s, 1H)

Preparation 6: 3-bromo-1H-pyrazol-5-amine

To a solution of 3,4,5-Tribromopyrazole (60 g) in Acetic acid (900 mL)at 10° C. was added Nitric acid (21 mL) (90%, fuming). Acetic anhydride(300 mL) was then added over 20 minutes. The reaction solution waswarmed to room temperature and stirred for 3 hours. The reaction mixturewas then poured over ice resulting in a white precipitate. Theprecipitate was filtered off and washed with water (200 mL). Thefiltered precipitate was then dissolved in Toluene (750 mL), washed withwater (200 mL) and brine (100 mL), dried over sodium sulfate, andfiltered. To the toluene solution was then added 1H-Pyrazole,3,5-dimethyl-(20 g), and the solution was heated at reflux for 20minutes. The reaction solution was cooled and concentrated in vacuo. Thecrude product was triturated with heptane and the resulting precipitatewhich contained mostly product by TLC was filtered and dried in vacuo.The crude title compound was carried on without further purification.(71.7 mg, 67.6%).

This intermediate, 3,4-dibromo-5-nitro-1H-pyrazole (69 g) was reduced byrefluxing with Stannous chloride, dihydrate (135 g) in Ethyl acetate(600 mL) and Ethanol (300 mL) at 110° C. for 45 minutes. The yellowhomogenous reaction solution was cooled to room temperature and slowlypoured over a vigorously stirring solution of sodium bicarbonate (33 g)in water (200 mL) and ethyl acetate (800 mL). To the resultant slurrywas added Celite (30 g), and this slurry was filtered through a bed ofCelite. The filter cake was washed with additional ethyl acetate (600mL). The organic solution was then washed with brine (200 mL), driedover sodium sulfate, filtered, and concentrated in vacuo to give thecrude product as an orange oil. The crude product was then purified byflash column chromatography (Biotage, Quad 25; Eluent: 6% EtOH inmethylene chloride). This afforded the title compound as a light beigesolid (13.2 g, 32%). ¹H NMR (400 MHz, DMSO-d6) δ: 5.20, (m, 3H), 11.60,(br s, 1H).

Preparation 7:2-bromo-5-(1-ethyl-3-methyl-1H-pyrazol-4-yl)-7-(trifluoromethyl)pyrazolo[1,5-a]pyrimidine

1-(1,3-dimethyl-1H-pyrazol-4-yl)-4,4,4-trifluoro-3-hydroxybut-2-en-1-one(Preparation 5, 1.7 g) and 3-bromo-1H-pyrazol-5-amine (Preparation 6,1.18 g) in Acetic acid (52.61 mL) was heated at 120° C. in a sealed tubeovernight. The reaction solution was cooled to room temperature, andpoured into ice water (500 mL) resulting in a white precipitate. Theprecipitate was filtered and washed with copious amounts of water. Theprecipitate was then collected and dried in vacuo to afford the titlecompound as a white powder (1.9 g, 73.1%). ¹H NMR (400 MHz, DMSO-d6) δ:2.47 (s, 3H), 3.79, (s, 3H), 6.95, (s, 1H), 7.73, (s, 1H), 8.67, (s,1H).

Preparation 8:2-bromo-5-(1,3-dimethyl-1H-pyrazol-4-yl)-7-(trifluoromethyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidine

Sodium tetrahydroborate (472 mg) was slowly added to a suspension of2-bromo-5-(1-ethyl-3-methyl-1H-pyrazol-4-yl)-7-(trifluoromethyl)pyrazolo[1,5-a]pyrimidine(Preparation 7, 1.8 g) in Ethanol (20 mL). The reaction mixture wasstirred at room temperature for 3 hours. The reaction solution was thenvery slowly added to ice cooled 1N HCl until a pH=2.0 was achieved toquench any remaining sodium tetrahydroborate. The solution was thenconcentrated under high vacuum to remove the majority of ethanol.Saturated sodium bicarbonate aqueous solution was then slowly added tothe acidic solution until a neutral pH (7.0) was achieved and a whiteprecipitate forms. The precipitate was filtered off and washed withwater (200 mL) and ether (20 mL). The white precipitate was collectedand dried in vacuo to afford the title compound as a white powder (1.36g, 74.7%). ¹H NMR (400 MHz, DMSO-d6) δ: 2.00, (m, 1H), 2.09, (s, 3H),2.30, (m, 1H), 3.67, (s, 3H), 4.34, (m, 1H), 5.15, (m, 1H), 5.31, (s,1H), 6.69, (s, 1H), 7.63, (s, 1H).

Preparation 9:(5S,7R)-2-bromo-5-(1,3-dimethyl-1H-pyrazol-4-yl)-7-(trifluoromethyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidine

The racemic2-bromo-5-(1,3-dimethyl-1H-pyrazol-4-yl)-7-(trifluoromethyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidine(1.5 g, 4.1 mmol, Preparation 8) was dissolved in methanol (35 mL) andthe cloudy solution was gently warmed providing a clear solution. Thesolution was then filtered through a medium porosity Buchner funnel. Theclear filtrate was directly used for chiral HPLC purification. 1 mL ofthis solution was loaded onto a 2.1 cm×25 cm Chiralcel OD column andeluted with a mobile phase comprising of isopropyl alcohol and methanol(1:1 ratio) at a flow rate of 15 mL/min. The two (R and S) enantiomerswere collected separately. 27 such injections were carried out and thepooled fractions of pure (R and S) enantiomers were concentrated underreduced pressure. The title compound was isolated as a white powder(0.71 g, 1.95 mmols, >95% ee).

Preparation 10:(5R,7S)-2-bromo-5-(1,3-dimethyl-1H-pyrazol-4-yl)-7-(trifluoromethyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidine

The racemic2-bromo-5-(1,3-dimethyl-1H-pyrazol-4-yl)-7-(trifluoromethyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidine(1.5 g, 4.1 mmol, Preparation 8) was dissolved in methanol (35 mL) andthe cloudy solution was gently warmed providing a clear solution. Thesolution was then filtered through a medium porosity Buchner funnel. Theclear filtrate was directly used for chiral HPLC purification. 1 mL ofthis solution was loaded onto a 2.1 cm×25 cm Chiralcel OD column andeluted with a mobile phase comprising of isopropyl alcohol and methanol(1:1 ratio) at a flow rate of 15 mL/min. The two (R and S) enantiomerswere collected separately. 27 such injections were carried out and thepooled fractions of pure (R and S) enantiomers were concentrated underreduced pressure. The title compound was isolated as a white powder(0.71 g, 1.95 mmols, >95% ee).

Example ER-889996

To a 5 mL screw-cap vial was added4-(Piperazine-1-carbonyl)phenylboronic acid, pinacol ester (51.0 mg,0.161 mmol), Tetrakis(triphenylphosphine)palladium(0) in 1,4-Dioxane(0.075M, 90 uL), a solution of2-bromo-5-(1,3-dimethyl-1H-pyrazol-4-yl)-7-(trifluoromethyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidine(25.0 mg, 0.069 mmol, Preparation 8) in 1,4-Dioxane (450 uL) and 2MSodium carbonate in Water (70 uL). The reactor vessel was purged withnitrogen and sealed. The vial was shaken and heated in an aluminum blockat 85° C. for 40 h. To the mixture was added 1.0 mL of saturated aqueoussodium bicarbonate and the mixture was then extracted with ethyl acetate(2×2.0 mL) The combined organic layers were concentrated in vacuo. Theremaining residue was purified by LC/MS according to method. Thisafforded the title compound as a white powder (7.8 mg, 24.0%).

Example ER-889862 was prepared from2-bromo-5-(1,3-dimethyl-1H-pyrazol-4-yl)-7-(trifluoromethyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidine(40.1 mg, 0.11 mmol, Preparation 8) and commercially availableIsoquinolin-5-yl boronic acid (2.5 eq) in a manner similar to that ofexample ER-889996. Purification by LCMS, using HPLC condition III,afforded the title compound as a white powder (7.5 mg, 17%).

Example ER-890007 was prepared from2-bromo-5-(1,3-dimethyl-1H-pyrazol-4-yl)-7-(trifluoromethyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidine(25.0 mg, 0.069 mmol, Preparation 8) and commercially available8-Methylquinolin-5-yl boronic acid (2.5 eq) in a manner similar to thatof example ER-889996. Purification by LCMS, using HPLC condition III,afforded the title compound as a white powder (12.2 mg, 42%).

Example ER-892900 was prepared from2-bromo-5-(1,3-dimethyl-1H-pyrazol-4-yl)-7-(trifluoromethyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidine(23.0 mg, 0.063 mmol, Preparation 8) and commercially available1,4-Dimethyl-1H-indazol-5-yl boronic acid (2.5 eq) in a manner similarto that of example ER-889996. Purification by LCMS, using HPLC conditionIII, afforded the title compound as a white powder (7.7 mg, 28%).

Example ER-890066 was prepared from2-bromo-5-(1,3-dimethyl-1H-pyrazol-4-yl)-7-(trifluoromethyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidine(25.0 mg, 0.069 mmol, Preparation 8) and commercially available2-Methyl-2H-indazol-5-yl boronic acid (2.5 eq) in a manner similar tothat of example ER-889996. Purification by LCMS, using HPLC conditionIII, afforded the title compound as a white powder (0.2 mg, 1%).

Example ER-889550

A sealed tube was charged with2-bromo-5-(1-ethyl-3-methyl-1H-pyrazol-4-yl)-7-(trifluoromethyl)pyrazolo[1,5-a]pyrimidine(40 mg, 0.11 mmols, Preparation 7),4-(N-Ethylaminocarbonyl)phenylboronic acid (73.2 mg, 0.38 mmols),Potassium carbonate (76.8 mg, 0.56 mmols), Water (0.559 mL), andTetrahydrofuran (0.838 mL). The reaction mixture was stirred while beingdegassed with nitrogen for 15 minutes.[1,1′-Bis(diphenylphosphino)ferrocene]dichloropalladium(II), complexwith dichloromethane (1:1) (22.7 mg, 0.03 mmols) was added and thereaction mixture was sealed and heated at 95° C. for 16 hours. The crudereaction mixture was cooled to room temperature and then purifieddirectly by flash column chromatography (Biotage Quad 25; Eluent: 100%EtOAc). This afforded4-(5-(1,3-dimethyl-1H-pyrazol-4-yl)-7-(trifluoromethyl)pyrazolo[1,5-a]pyrimidin-2-yl)-N-ethylbenzamide as a light yellow solid (41.1 mg, 86.4%).

Sodium tetrahydroborate (6.62 mg) was added to a suspension of4-(5-(1,3-dimethyl-1H-pyrazol-4-yl)-7-(trifluoromethyl)pyrazolo[1,5-a]pyrimidin-2-yl)-N-ethylbenzamide (30 mg, 0.07 mmols) in ethanol (1 mL) and the mixture stirred atroom temperature for 3 hours. The reaction solution was concentratedunder a stream of nitrogen. 1N HCl was then very slowly added until theborohydride was fully consumed (˜2 mL) resulting in a brown precipitate.The aqueous solution was decanted off and slowly neutralized withsaturated sodium bicarbonate until pH=7.0 was reached. The neutralizedsolution was then extracted with EtOAc (2×5 mL). The organic extractswere combined and washed with brine (2 mL), dried over sodium sulfate,filtered and concentrated in vacuo. This afforded the title compound asa white powder (18.8 mg, 62.1%).

Example ER-893888

A sealed tube was charged with2-bromo-5-(1,3-dimethyl-1H-pyrazol-4-yl)-7-(trifluoromethyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidine(30 mg, 0.08 mmols, Preparation 8), 4-(Ureido)phenylboronic acid,pinacol ester (73.8 mg, 0.28 mmols), Potassium carbonate (56.9 mg, 0.41mmols), Water (0.414 mL), and Tetrahydrofuran (0.621 mL) The reactionmixture was stirred while being degassed with nitrogen for 15 minutes.[1,1′-Bis(diphenylphosphino)ferrocene]dichloropalladium(II), complexwith dichloromethane (1:1) (16.8 mg, 0.02 mmols) was added and thereaction mixture was sealed and heated at 95° C. for 16 hours. The crudereaction mixture was cooled to room temperature and then purifieddirectly by flash column chromatography (Biotage Quad 25; Eluent: 10%EtOH in EtOAc). This afforded the title compound as a light yellowpowder (31.1 mg, 90.0%).

Example ER-894595 was prepared from(5S,7R)-2-bromo-5-(1,3-dimethyl-1H-pyrazol-4-yl)-7-(trifluoromethyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidine(150.0 mg, 0.41 mmol, Preparation 9) and commercially available4-(Ureido)phenylboronic acid, pinacol ester (368.8 mg, 1.4 mmols) in amanner similar to that of example ER-893888. Purification by flashcolumn chromatography (Biotage Quad 25; Eluent: 10% EtOH in EtOAc)afforded the title compound as a light yellow powder (141.6 mg, 82.0%).

Example ER-894596 was prepared from(5R,7S)-2-bromo-5-(1,3-dimethyl-1H-pyrazol-4-yl)-7-(trifluoromethyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidine(150.0 mg, 0.41 mmol, Preparation 10) and commercially available4-(Ureido)phenylboronic acid, pinacol ester (368.8 mg, 1.4 mmols) in amanner similar to that of example ER-893888. Purification by flashcolumn chromatography (Biotage Quad 25; Eluent: 10% EtOH in EtOAc)afforded the title compound as a light yellow powder (144.6 mg, 83.7%).

Example ER-893986 was prepared from(5S,7R)-2-bromo-5-(1,3-dimethyl-1H-pyrazol-4-yl)-7-(trifluoromethyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidine(50.0 mg, 0.14 mmol, Preparation 9) and commercially available1,4-Dimethyl-1H-indazole-5-boronic acid (89.1 mg, 0.47 mmols) in amanner similar to that of example ER-893888. Purification by flashcolumn chromatography (Biotage Quad 25; Eluent: 10% EtOH in EtOAc)afforded the title compound as a light yellow powder (42.7 mg, 72.4%).

Example ER-893987 was prepared from(5R,7S)-2-bromo-5-(1,3-dimethyl-1H-pyrazol-4-yl)-7-(trifluoromethyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidine(150.0 mg, 0.41 mmol, Preparation 10) and commercially available1,4-Dimethyl-1H-indazole-5-boronic acid (267.3 mg, 1.41 mmols) in amanner similar to that of example ER-893888. Purification by flashcolumn chromatography (Biotage Quad 25; Eluent: 10% EtOH in EtOAc)afforded the title compound as a light yellow powder (155.7 mg, 88.0%).

Example ER-893990 was prepared from(5S,7R)-2-bromo-5-(1,3-dimethyl-1H-pyrazol-4-yl)-7-(trifluoromethyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidine(40.0 mg, 0.11 mmol, Preparation 9) and commercially available2-Methylindazole-5-boronic acid pinacol ester (96.8 mg, 0.38 mmols) in amanner similar to that of example ER-893888. Purification by flashcolumn chromatography (Biotage Quad 25; Eluent: 10% EtOH in EtOAc)afforded the title compound as a light yellow powder (36.9 mg, 80.9%).

Example ER-893991 was prepared from(5R,7S)-2-bromo-5-(1,3-dimethyl-1H-pyrazol-4-yl)-7-(trifluoromethyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidine(30.0 mg, 0.082 mmol, Preparation 10) and commercially available2-Methylindazole-5-boronic acid pinacol ester (72.6 mg, 0.28 mmols) in amanner similar to that of example ER-893888. Purification by flashcolumn chromatography (Biotage Quad 25; Eluent: 10% EtOH in EtOAc)afforded the title compound as a light yellow powder (22.0 mg, 64.3%).

Synthetic Examples Section J

A suspension of methyl 5-amino-1H-pyrazole-3-carboxylate (1.00 g, 7.086mmol) and 1-(3,4-dimethoxyphenyl)-4,4,4-trifluorobutane-1,3-dione (2.153g, 7.794 mmol) in ACETIC ACID (10 ml, 174.682 mmol) was heated to reflux(at 100° C.) for 5 hours. The mixture was cooled at room temperature,water was added and the precipitate collected by filtration, washed withwater and dried under vacuum to give J1 as a green solid, 2.33 g (86%yield).

Sodium borohydride (0.446 g, 11.802 mmol) was slowly added to asuspension of methyl5-(3,4-dimethoxyphenyl)-7-(trifluoromethyl)pyrazolo[1,5-a]pyrimidine-2-carboxylate(1.00 g, 2.623 mmol) in ETHANOL (25 ml, 428.167 mmol) at roomtemperature. The mixture was heated to reflux (80° C.) for 60 minutesand reaction was monitored by UPLC/MS. The mixture was cooled at roomtemperature. The excess sodium borohydride was decomposed with ACETICACID (1.501 ml, 26.226 mmol). The reaction was poured into 1.00 M HCl inWater (52.5 ml, 52.451 mmol) and was stirred for 5 minutes. Theprecipitate was collected by filtration, washed with water and airdried. The material was purified by flash chromatography using a 25 gsilica column with a heptane:ethyl acetate gradient to give J2 as awhite solid, 507 mg (48% yield).

To a suspension of ethyl5-(3,4-dimethoxyphenyl)-7-(trifluoromethyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidine-2-carboxylate(503 mg, 1.26 mmol) in ethanol (5.0 mL, 85.633 mmol) was added 4.00 Msodium Hydroxide in water (1.574 mL, 6.298 mmol). The mixture was heatedat 50° C. for 1 hour. The mixture was cooled at room temperature and wasacidified with 1.00 M HCl in Water (12.60 mL, 12.595 mmol), water wasadded and the precipitate collected by filtration, washed with water anddried under vacuum to give J3 as a white solid, 422 mg (95% yield).

A 5 mL microwave vial was charged with5-(3,4-dimethoxyphenyl)-7-(trifluoromethyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidine-2-carboxylicacid (50 mg, 0.135 mmol), tert-butyl piperazine-1-carboxylate (75 mg,0.404 mmol), HATU (205 mg, 0.539 mmol), N,N-diisopropylethylamine (0.059mL, 0.337 mmol) and DMF (1.0 mL, 12.915 mmol). The vial was capped andthe mixture was stirred for 6 hours at 40° C. The mixture was dilutedwith Ethyl acetate (5 mL), washed 2× with water (2 mL), washed with 0.1Naqueous HCl (2 mL), washed with saturated aqueous sodium bicarbonate (2mL), washed with brine (2 mL), dried with sodium sulfate, filtered andconcentrated. Compound was purified by chromatography on Biotage SP4(Column Interchim 25 g, 30 μM) using 12-100% EtOAc/Heptane as eluent toafford intermediate tert-butyl4-(5-(3,4-dimethoxyphenyl)-7-(trifluoromethyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidine-2-carbonyl)piperazine-1-carboxylate(38 mg, 0.070 mmol, 52.3% yield).

The intermediate tert-butyl4-(5-(3,4-dimethoxyphenyl)-7-(trifluoromethyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidine-2-carbonyl)piperazine-1-carboxylate(36 mg, 0.067 mmol) was dissolved in ethanol (0.5 mL, 8.563 mmol) and4.00M HCl in Dioxane (0.5 mL, 2.00 mmol) and the mixture was stirred at40° C. for 1 hour. The reaction mixture was concentrated and the productwas dried on vacuum pump to afford ER-896452 as the HCl salt (31.0 mg,0.065 mmol, 98% yield).

ER-896453

Example ER-896453 was prepared in two steps from acid J3 (50 mg, 0.135mmol) and commercially available (S)-tert-butyl3-aminopiperidine-1-carboxylate (81 mg, 0.404 mmol) in a manner similarto that of example ER-896452 to afford intermediate (3S)-tert-butyl3(5-(3,4-dimethoxyphenyl)-7-(trifluoromethyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidine-2-carboxamido)piperidine-1-carboxylate(67 mg, 0.121 mmol, 90% yield). This intermediate (49 mg, 0.089 mmol)was treated with HCl in a manner similar to that of example ER-896452 toafford the desired product ER-896453 (42 mg, 97% yield).

Determination of Absolute Configuration of ER-885454 and its Correlationto ER-892887

The absolute configuration of ER-885454 was established by converting itto the 3-iodo derivative ER-887006 as shown above. The structure ofER-887006 was determined by single crystal X-ray diffraction. Resultsare shown in FIG. 10. Since this transformation does not affect thechiral centers in the molecule, the absolute configuration of ER-885454is as shown. Moreover, the absolute configuration of ER-892887 wasestablished via its correlation to compound C-6, which in turn wascorrelated to ER-885454.

General Screening Assay and Pharmacology Strategy.

To identify potent and selective TLR7/8 compounds, analogs wereinitially screened across a cell-based panel of human TLR4, TLR7, andTLR9 reporter lines (see Materials and Methods for more details). Asubset of compounds that were potent and selective for TLR7 were alsotested for TLR8 activity (see Table 2 below) and for TLR7/8 potency inthe primary human PBMC assay (see Materials and Methods for moredetails). Certain compounds were advanced into the short-term in vivo(STIV) assay to determine dose-dependent activity and duration-of-actionagainst mouse TLR7 (see Materials and Methods for more details). Selectcompounds were then evaluated for impact in one or more of the followingmouse lupus disease models: BXSB-Yaa, NZBxNZW, and Pristane:DBA/1.

Many compounds reported as embodiments herein demonstrate nanomolarpotency against both human and mouse TLR7 and human TLR8 when thesereceptors, expressed on either cell lines or primary cells, arestimulated by synthetic, small molecule (CLO97, R848) or nucleic-acid(RNA) ligands. Conversely, most compounds reported as embodiments hereinare inactive against the TLR9 pathway.

Current lupus SOC drugs include anti-malarials such as chloroquine andhydroxychloroquine (HCQ) which have been shown to inhibit TLR7/9signaling in vitro. This may at least partially explain theireffectiveness in controlling lupus flare. Embodiments of the disclosure,however, have been shown to offer significantly more potent inhibition.For example, compound ER-892887 (shown and discussed above) was found tobe approximately 100-fold more potent against the RNA-Ig TLR7/8 stimulusversus HCQ (ER-892887 IC₅₀=0.015 uM, HCQ IC₅₀˜1.5 uM). This suggeststhat ER-892887 would offer much more effective TLR7/8 pathway inhibitionversus current lupus treatments. This is demonstrated by results shownin Table 1 below.

TABLE 1 Potency and selectivity of compound ER-892887 as compared tohydroxychloroquine (Plaquenil ®). Cell ER-892887 HCQ² Format: Ligand:Receptor(s): Analyte: IC50 (uM) IC50 (uM) HEK-293 LPS Human TLR4NFkB-luciferase >10 N.D. HEK-293 CLO97 Human TLR7 NFkB-luciferase 0.010N.D. HEK-293 CLO97 Mouse TLR7 NFkB-luciferase 0.276 N.D. HEK-293 CLO97Human TLR8 NFkB-luciferase 0.080 N.D. HEK-293 CpG-ODN Human TLR9NFkB-luciferase >10 N.D. Hu PBMC ¹RNA-Ig Human TLR7/8 IL-6 0.015 1-2 HuPBMC ¹RNA-Ig Human TLR7/8 TNFα 0.013 N.D. Hu PBMC ¹RNA-Ig Human TLR7/8IP-10 0.045 N.D. Hu PBMC R848 Human TLR7/8 IL-6 0.016 N.D. Mu SpleenR848 Mouse TLR7 IL-6 0.070 N.D. Hu PBMC Pam3CSK4 Human TLR1/2 IL-6 >10N.D. Hu PBMC LPS Human TLR4 IL-6 >10 >10 Hu PBMC CpG-ODN Human TLR9IL-6 >10 0.15-0.30 TABLE KEY: ¹RNA-Ig = ssRNA derived from U1snRNA stemloop IV sequence in complex with antibody (see Materials and Methods formore details) ²HCQ = Hydroxychloroquine

TABLE 2 Potency of select compounds against human TLR8 in the HEK-293assay format (see Materials and Methods for more details). HEK/TLR8ER-number IC50 (μM) ER-885454-00 0.28 ER-885484-00 >10 ER-885690-00 1.49ER-886434-00 0.30 ER-886622-00 0.07 ER-887534-00 0.41 ER-887734-00 0.14ER-887738-00 >10 ER-892887-00 0.08 ER-892889-00 0.15 ER-892890-00 1.47ER-892892-00 0.14 ER-892900-00 0.33 ER-892924-00 0.22 ER-893888-00 0.46ER-893961-00 0.16 ER-893969-00 0.25 ER-893972-00 0.30 ER-893987-00 0.22ER-894595-00 0.32 ER-894596-00 0.54

Short-Term In Vivo (STIV) Assay:

To assess compound potency in vivo against mouse TLR7, a short-term invivo (STIV) assay was utilized. Briefly, mice were orally dosed withcompounds and at various time points afterwards were injectedsubcutaneously with agonist R848 to stimulate TLR7. The plasma IL-6level following R848 stimulation was then measured by ELISA to assesscompound potency and duration-of-action. Importantly, cytokineproduction following in vitro or in vivo stimulation with R848 was shownto be completely TLR7-dependent utilizing TLR7-deficient mice.Therefore, the activity of compounds in the STIV assay can beconfidently attributed to their modulation of the TLR7 pathway. A singleoral dose of ER-892887 at 300 mg/kg suppresses the R848/TLR7/IL-6pathway in vivo by 80-90% for at least 24 hours (see FIG. 1). A summaryof STIV assay potency for a panel of compounds appears in Table 3 below.

TABLE 3 Short-term in vivo (STIV) assay data summary for selectcompounds. Short-term In Vivo (STIV) Assay Data Summary Time Dose %Suppression vs. Vehicle Point: (mg/kg) ER-892887 ER-892893 ER-889549ER-892889 ER-892890 ER-889904 ER-893972  3 hr 11 33 100 30 93 98 64 9883 300  6 hr 11 33 83 23 43 48 68 77 83 100 100 0 84 99 84 100 96 300 13hr 11 27 33 12 18 78 100 49 6 92 300 90 19 hr 11 33 7 53 100 0 0 45 24300 24 hr 11 33 0 100 12 300 86 Time Dose % Suppression vs. VehiclePoint: (mg/kg) ER-892892 ER-885681 ER-893971 ER-895676 ER-895678ER-895498 ER-895496  3 hr 11 33 90 100 96 98 99 300 99  6 hr 11 33 86 4299 0 0 35 71 100 94 81 99 300 96 13 hr 11 33 0 10 0 0 34 33 100 7 99 2828 56 43 300 50 0 8 81 96 19 hr 11 33 49 100 52 300 24 hr 11 33 0 25 35100 0 0 39 13 300 27 3 70 45 Time Dose % Suppression vs. Vehicle Point:(mg/kg) ER-895302 ER-895303 ER-894680 ER-885454 ER-894595  3 hr 11 33100 98 300  6 hr 11 33 93 92 98 0 100 99 98 57 300 99 13 hr 11 33 14 0 40 100 16 18 95 0 300 91 90 19 hr 11 33 100 56 300 100 24 hr 11 33 30 1000 0 300 37 60

Mouse Lupus Disease Models.

Three distinct lupus disease models (BXSB-Yaa, NZBxNZW, and Pristane)were chosen for compound POC evaluation because (1) the BXSB-Yaa andNZB/W strains develop spontaneous disease with polygenic etiology,demonstrating many hallmarks of human lupus such as DNA- andRNA-associated autoreactivity, proteinuria, and immune-complex mediatednephritis, and (2) positive TLR7 and/or TLR9 target validation resultshave been reported for all three disease models.

Key findings for ER-892887 in the SLE disease models are as follows (seeFIG. 1, FIG. 2A-FIG. 2D, FIG. 3A-FIG. 3E, FIG. 4A-FIG. 4F, FIG. 5A-FIG.5D, FIG. 6A-FIG. 6G, FIG. 12A-FIG. 12F, FIG. 13A-FIG. 13E, and FIG.14A-FIG. 14C):

-   1) ER-892887 significantly reduced anti-dsDNA titers in the    TLR7-driven BXSB-Yaa model, corresponding to a trend towards reduced    proteinuria and complete prevention of Grade 3/4 nephritis onset.-   2) ER-892887 reduced anti-dsDNA titers in the NZB/W model. Lower    doses (33-100 mg/kg) of ER-892887 afforded survival benefit in this    model, corresponding to reduced proteinuria and histological signs    of glomerulonephritis. ER-892887 delayed the development of    proteinuria in the NZB/W model more effectively than two drugs    commonly used in the treatment of human lupus, and was efficacious    in combination with hydroxychloroquine and prednisolone.-   3) ER-892887 suppressed various auto-antibodies in the Pristane    model, with particularly robust impact on RNA-related reactivity    such as anti-RiboP titers. These changes corresponded to a reduction    of whole blood interferon-driven gene expression and IFN gene    signature score by ER-892887 in this model. ER-892887 reduced    arthritis severity in the pristane model, and suppressed the    interferon gene signature score significantly, while three commonly    used lupus treatments did not. ER-892887 also suppressed    autoantibodies better than hydroxychloroquine or prednisolone at the    doses tested.

Key findings for ER-885454 in the SLE disease models are as follows (seeFIG. 7A-FIG. 7D, FIG. 8A-FIG. 8D, and FIG. 9A-FIG. 9C):

-   1) ER-885454 significantly reduced anti-dsDNA and anti-Sm/nRNP    titers in the TLR7-driven BXSB-Yaa model, corresponding to    significantly reduced proteinuria and complete prevention of Grade    3/4 nephritis onset.-   2) ER-885454 reduced anti-dsDNA, proteinuria, and histological signs    of glomerulonephritis in the NZB/W model.

Summary of Findings:

These data show a moderating effect of the compounds described onprocesses involved in important aspects of human lupus. Immune complexescontaining nucleic acids can drive type 1 interferon production bydendritic cells, and the “interferon signature”, reflecting presence ofinterferon and subsequent expression of interferon regulated genes, isassociated with disease severity. ER-892887 blocks cytokine responses toRNA-Ig complexes in vitro, and suppressed the upregulation ofinterferon-driven genes in the pristane model. Both ER-892887 andER-885454 limited the production of several autoantibody specificities,and suppressed kidney disease as manifested by proteinuria andhistologic changes. Treatment with ER-892887 significantly enhancedsurvival in long-term dosing studies in the spontaneous NZB/W model. Theresults indicate that these compounds have the potential to controllupus symptoms and progression in human patients.

Pharmacology Materials & Methods:

In vitro pharmacology: HEK-293 cells (ATCC) were engineered to stablyexpress a NF-kappaB transcription factor inducible E-selectin (ELAM-1)luciferase reporter derived from the plasmid pGL3 (Promega) containingbase pairs −2241 bp to −254 bp from the promoter of the human E-selectingene (Accession No. NM_000450). These cells were then subsequentlyengineered to stably and individually express human TLR4, TLR7 or TLR9full-length ORF cDNAs. Human TLR4 cDNA (Accession No. NM_138554) wascloned into pcDNA 3.0 expression vector (Invitrogen). TLR4 transfectedcells were also engineered to express human MD-2 co-receptor [MD-2 cDNA(Accession No. NM_015364) was cloned into the pEF-BOS vector] and weresupplemented with 10 nM soluble CD14 (R&D Systems) in the media tooptimize LPS responsiveness. Human TLR9 cDNA (Accession No. NM_017442)was cloned into the pBluescript II KS vector (Agilent). Human TLR7 cDNA(Accession No. NM_016562) was obtained from OriGene. HEK-293 cellsstably expressing human TLR8 (Accession No. NM_138636) or mouse TLR7(Accession No. NM_133211) were purchased from InvivoGen and were thenstably transfected with pNiFty2(NF-kappaB)-luciferase reporter plasmid(InvivoGen). Each cell type was plated in Dulbecco's modified Eagle'smedium (DMEM) containing 10% fetal bovine serum (FBS) at a density of2.22×10⁵ cells/ml into a 384-well plate and incubated for 2 days at 37°C., 5% CO₂. Varying concentrations of antagonist compounds were thenadded. Cells were then incubated for another 30 minutes before addingthe appropriate TLR agonist as follows (final concentrations indicated):lipopolysaccharide (LPS; Sigma) at 10 ng/ml for TLR4, CLO97 (InvivoGen)at 3 ug/ml for human TLR7 and TLR8 and mouse TLR7, and CpG-2006-2A[sequence: TCGTCGTTAAGTCGTTAAGTCGTT (SEQ ID NO: 1) with phosphorothioatebackbone, synthesized by Sigma-Aldrich] at 0.6 uM for TLR9. The cellswere then incubated overnight, and NF-kappaB dependent luciferasereporter activation was quantified by measuring luminescence withSteadyGlo® (Promega) or Steadylite™ (Perkin Elmer) reagent as per themanufacturer's suggested protocol.

Human PBMC Cell-Based Assay.

Human peripheral blood mononuclear cells (PBMC) were isolated fromfreshly-drawn heparinized (10 USP units/ml, Hospira, Lakeforest, Ill.)healthy donor whole blood by density gradient (Histopaque® 1077, Sigma,Inc., St. Louis, Mo.). Briefly, 25 ml blood was diluted with 15 ml PBS(without Ca²⁺, Mg²⁺) in a 50 ml conical tube, and 12 ml Histopaque wasunderlaid using a spinal needle. Tubes were centrifuged for 45 minutesat 1200 rpm (350×g), and PBMC were collected from the buffy coat. Cellswere then washed twice in PBS, and red blood cells were lysed bysuspension in 5 ml ammonium chloride solution (1× Red Blood Cell LysisBuffer, eBioscience) for 5 minutes at room temperature. After a finalwash in PBS, PBMC were resuspended at a final concentration of 2×10⁶/mlin RPMI-1640 media with L-glutamine (Invitrogen) and supplemented with25 mM HEPES (Mediatech, Inc, Manassas Va.), 10% fetal bovine serum(HyClone, Logan, Utah), and Penicillin-Streptomycin-Glutamine(Mediatech) and plated at 100 ul/well (2×10⁵ cells/well) in tissueculture treated 96-well plates (Falcon).

Antagonist compounds solubilized and serial diluted in 100% DMSO wereadded in triplicate to cells to yield a final concentration of 0.1% DMSO(v/v). Hydroxychloroquine (Acros Organics) solubilized and serialdiluted in PBS was added in triplicate to cells. PBMC were incubatedwith antagonist compounds or HCQ for 30 minutes at 37° C., 5% CO₂ beforeadding various TLR agonist reagents in 100 ul complete media per well asfollows (final concentrations indicated): R848 (Resiquimod; GLSynthesis,Worcester, Mass.) at 1 uM for TLR7 and TLR8, Pam3CSK4 (InvivoGen) at 50ng/ml for TLR1/2, LPS (Sigma) at 10 ng/ml for TLR4, and CpG-2216(InvivoGen) at 5 ug/ml for TLR9. To prepare a TLR7/8 agonist that mimicsRNA-containing auto-antibody immune complexes in lupus patients, a26-mer RNA with a sequence derived from human U1 snRNA stem loop IV[(sequence: GGGGGACUGCGU-UCGCGCUUUCCC (SEQ ID NO: 2) withphosphorothioate backbone] was synthesized (Dharmacon, Inc., Lafayette,Colo.), which has been shown previously to be a potent TLR7 and TLR8agonist. This RNA molecule was diluted to 2.5 μM in serum-free RPMI, andmouse anti-human single stranded DNA monoclonal antibody (MAB3034,Millipore, Inc., Billerica, Mass.), which also cross-reacts with RNA,was added at a 1:25 dilution or at 1 ug/ml. The resulting “RNA-Ig”stimulus was incubated at room temperature for 15-30 minutes beforeadding to cells. PBMC were incubated with the various TLR agonists for20 hours at 37° C., 5% CO₂. Cell culture supernatants were collected,and levels of various human cytokines were assessed as indicated bystandard ELISA procedure according to the manufacturer's recommendedprotocol (BD Biosciences, Inc., San Diego, Calif.).

Mouse Spleen Cell-Based Assay.

Spleens were harvested from female BALB/c mice (Jackson Labs, BarHarbor, Me.) euthanized by CO₂. A single cell suspension was obtained bypassing spleens through a 40 μm nylon cell strainer. Cells were washedtwice with 50 ml PBS (Mediatech, Inc., Manassas, Va.) and red bloodcells were lysed in 5 ml RBC Lysis buffer (eBioscience, Inc., San Diego,Calif.) for 5 minutes at room temperature. Cells were washed twice morein PBS and finally resuspended in supplemented RPMI-1640 at 2.5×10⁶cells/ml. Cells were plated at 100 μl/well (2.5×10⁵ cells/well) in96-well tissue culture treated plates (Falcon). Serial dilutions ofcompounds solubilized in 100% DMSO were added in triplicate to cells toyield a final concentration of 0.1% DMSO. Cells were incubated withcompound for 30 minutes at 37° C., 5% CO₂ before adding 100 μl/well of740 nM R848 (Resiquimod; GLSynthesis, Worcester, Mass.) in completemedia for a final concentration of 370 nM R848. Cells were incubated for20 hours at 37° C., 5% CO₂. Culture supernatants were collected, andlevels of IL-6 were assessed by standard ELISA procedure according tothe manufacturer's recommended protocol (BD Biosciences, Inc., SanDiego, Calif.).

In vivo pharmacology:

Short-Term In Vivo (STIV) Assay.

Six to eight week old female BALB/c mice (Jackson Labs, Bar Harbor, Me.)were dosed by oral gavage in 200 ul volume with antagonist compoundsformulated in 0.5% aqueous methyl-cellulose (Sigma, St. Louis, Mo.). Atvarious time points afterwards, mice were injected subcutaneously (s.c.)in 100 ul volume with 15 ug R848 (Resiquimod; GLSynthesis, Worcester,Mass.) to stimulate TLR7. Blood plasma was collected by cardiacpuncture, and levels of IL-6 at 1.5 hours after TLR7 stimulation werethen assessed by standard ELISA procedure according to themanufacturer's recommended protocol (R&D Systems).

Mouse Lupus Disease Model Strains.

Male BXSB-Yaa and female NZBWF1/J mice were purchased from Jackson Labs(Bar Harbor, Me.), both of which manifest with spontaneous lupusdisease. Female DBA/1 mice were purchased from Harlan Laboratories(Indianapolis, Ind.) and at the indicated ages given an intraperitonealinjection of 0.5 ml pristane (2,6,10,14-Tetramethylpentadecane; Sigma,St. Louis, Mo.) to chemically induce lupus disease or of 0.5 ml PBS togenerate age-matched, non-diseased control mice. Mice were dosed dailyby oral administration of compound or drug in 0.5% methylcellulose, forthe time period indicated.

Assessment of Auto-Antibody Titers by ELISA.

Anti-dsDNA, -Sm/nRNP, -RiboP, and Histone titers were evaluated bystandard ELISA approach. Briefly, 96-well EIA/RIA ELISA plates (Corning)were coated with 100 ul of diluted antigen in PBS for 90 minutes at roomtemperature as follows (final concentrations indicated): 10 U/ml Sm/nRNPcomplex (Immunovision), 10 ug/ml calf thymus dsDNA (Sigma), 5 U/ml RiboP(Immunovision), and 5 ug/ml Histone (Immunovision). Plates were washedwith PBS/0.05% Tween20 (washing buffer) and blocked overnight withPBS/1% BSA (blocking buffer) at 4 C. Plates were washed, mouse plasmasamples diluted in blocking buffer (ranging from 1:25-1:10,000 dependingon the model and the antigen) were added to wells in 100 ul volume perwell, and plates were incubated for 90 minutes at room temperature.Plates were then washed, 100 ul anti-mouse-IgG-HRPO (Southern Biotech)diluted 1:50,000 in PBS/1% BSA/0.05% Tween was added to each well, andplates were incubated for 90 minutes at room temperature. Plates werewashed, and 100 ul of a 1:1 mix of substrate components from the OptEIATMB substrate kit (BD Biosciences) was added to the wells. Plates wereincubated at room temperature, and after sufficient color developmentthe reaction was stopped by adding 100 ul of 0.18M sulfuric acidsolution. Plates were read by spectrophotometry at 450 nm.

Assessment of Proteinuria.

Urine was collected manually from individual mice or by housing 1-2 miceper metabolic cage for 18 hours, and the Urinary Albumin CreatinineRatio (UACR) was determined for each animal as an indirect measure ofkidney function (UACR calculated as the ratio of mg of albumin/g ofcreatinine per dL of urine). Albumin levels in the urine samples weredetermined using a custom sandwich ELISA protocol using an anti-mousealbumin antibody set (Bethyl Labs), which included a coating antibodyand a secondary antibody tagged with an HRP conjugate for detection.Creatinine levels were determined using a commercial creatinine assaykit (Cayman).

Histological Assessment of Nephritis.

Kidneys were collected from individual mice, fixed in 10% formalin for24 hours, embedded in paraffin, and H&E stained sections were generatedfor histopathology assessment in a blinded fashion. Features ofNephritis Disease Scores are as follows: Grade 0—normal limits; Grade1—ribbon-like capillary wall thickening; Grade 2—hypercellularity,segmentation, crescent formation; Grade 3—see Grade 2, increasedseverity and extent (% glomeruli affected) of glomerular lesions; Grade4—sclerosis; severe glomerular disease (non-functional organ).

Statistics:

Differences in UACR, cytokine or antibody titer between drug-treated andvehicle-treated groups were calculated using values from all individualanimals in the group. They were tested by one-way ANOVA with Dunn'spost-test to compare each experimental group to vehicle. P values arestated in the figures, or by convention a single asterisk indicatesp<0.05, two asterisks indicates p<0.01 and three asterisks indicatesp<0.001. Sets of mortality curves were compared by Mantel-Cox, and wheresignificance was found, pairs of curves were tested for difference usingWilcoxon analysis.

Assessment of Interferon Gene Expression in Whole Blood.

The expression of IFN-regulated genes in whole blood was measured byqPCR. Briefly, mice were euthanized, blood was collected via the venacava, and 100 ul was preserved in tubes containing RNAlater (Ambion,Austin Tex.). Total RNA was isolated using the Mouse RiboPure Blood RNAIsolation Kit (Ambion). RNA concentrations were determined using aNanoDrop ND-1000 spectrophotometer (Thermo Scientific, Waltham Mass.).First strand cDNA was synthesized from 100 ng total RNA using HighCapacity RNA-to-cDNA Master Mix (Applied Biosystems, Foster CityCalif.). After reverse transcription, cDNA was diluted withnuclease-free water and mixed with Gene Expression Master Mix (AppliedBiosystems). The mixture was then applied to a custom TaqMan® LowDensity Array (TLDA) manufactured by Applied Biosystems, and qPCR wasperformed on the ABI 7900HT Fast Real-time PCR System (AppliedBiosystems). Raw data was collected using RQ Manager 1.2.1 (AppliedBiosystems) and analyzed using GeneData Analyst 2.2 software (GeneData).

The TLDA panel contained as many as 45 target genes and 3 housekeepinggenes for normalization (see Table 4 below). The housekeeping gene Hprt1was chosen for normalization based on coefficient-of-variation. Relativequantities were determined for the target genes and used to calculate afold change for each diseased mouse relative to the non-diseased controlgroup receiving intraperitoneal PBS injection only. A standard Student'st-test was performed to determine which target genes were significantlyincreased between the non-diseased group (PBS treated) and thevehicle-treated diseased group (pristane treated), thereby representingthe disease-regulated gene set. An “IFN score” was subsequentlycalculated for each mouse as the median fold change of alldisease-regulated genes identified in the t-test.

TABLE 4 Gene symbol Taqman ID Gene name 18S Hs99999901_s1 Eukaryotic 18SrRNA Bst2 Mm01609165_g1 bone marrow stromal cell antigen 2 C1qaMm00432142_m1 Complement component 1q C3 Mm00437858_m1 complementcomponent 3 C3ar1 Mm02620006_s1 complement component 3a receptor 1 Ccl2Mm00441243_g1 chemokine (C-C motif) ligand 2 Ccr2 Mm00438270_m1chemokine (C-C motif) receptor 2 Cd300e Mm00468131_m1 CD300e antigenCMPK2 Mm00469582_m1 cytidine monophosphate (UMP-CMP) kinase 2 CD38Mm01220906_m1 CD38 antigen Cxcl10 Mm00445235_m1 chemokine (C-X-C motif)ligand 10 DDX60 Mm00460708_m1 DEAD (Asp-Glu-Ala-Asp) box polypeptide 60Elane Mm00469310_m1 elastase, neutrophil expressed Epsti1 Mm00712734_m1epithelial stromal interaction 1 (breast) Fcgr1 Mm00438874_m1 Fcreceptor, IgG, high affinity I Fpr1 Mm00442803_s1 formyl peptidereceptor 1 Gapdh Mm99999915_g1 glyceraldehyde-3-phosphate dehydrogenaseHERC6 Mm01341950_m1 hect domain and RLD 6 Hprt Mm00446968_m1hypoxanthine guanine phosphoribosyl transferase Ifi202b Mm00839397_m1interferon activated gene 202B Ifi204 Mm00492602_m1 interferon activatedgene 204 IFI27 Mm01329883_gH interferon, alpha-inducible protein 27 like2A Ifi35 Mm00510329_m1 interferon-induced protein 35 Ifi44 Mm00505670_m1interferon-induced protein 44 IFIH1 Mm00459183_m1 erferon induced withhelicase C domain 1 Ifit1 Mm00515153_m1 interferon-induced protein withtetratricopeptide repeats 1 IFIT2 Mm00492606_m1 interferon-inducedprotein with tetratricopeptide repeats 2 IFIT3 Mm01704846_s1interferon-induced protein with tetratricopeptide repeats 3 Irf7Mm00516788_m1 interferon regulatory factor 7 Isg15 Mm01705338_s1 ISG15ubiquitin-like modifier Isg20 Mm00469585_m1 interferon-stimulatedprotein Ly6e Mm01200460_g1 lymphocyte antigen 6 complex, locus E Mmp8Mm00439509_m1 matrix metallopeptidase 8 Mmp9 Mm00442991_m1 matrixmetallopeptidase 9 Mpo Mm00447886_m1 myeloperoxidase Ms4a6cMm00459296_m1 membrane-spanning 4-domains, subfamily A, member 6C Mx1Mm00487796_m1 myxovirus (influenza virus) resistance 1 Oas3Mm00460944_m1 2-5 oligoadenylate synthetase 3 Oasl2 Mm00496187_m1 2-5oligoadenylate synthetase-like 2 Ppia Mm02342430_g1 peptidylprolylisomerase A (cyclophilin A) Rsad2 Mm00491265_m1 radical S-adenosylmethionine domain containing 2 SIGLEC1 Mm00488332_m1 sialic acid bindingIg-like lectin 1, sialoadhesin Stat1 Mm00439531_m1 signal transducer andactivator of transcription 1 Tlr7 Mm00446590_m1 toll-like receptor 7Tlr9 Mm00446193_m1 toll-like receptor 9 Tnf Mm00443258_m1 tumor necrosisfactor Tnfsf13b Mm00446347_m1 tumor necrosis factor (ligand)superfamily, member 13b Treml4 Mm00553947_m1 triggering receptorexpressed on myeloid cells-like 4 Usp18 Mm00449455_m1 ubiquitin specificpeptidase 18 Xaf1 Mm01248390_m1 XIAP associated factor 1

We claim:
 1. A method for treatment of a systematic lupus erythematosusor lupus, comprising administering a pharmaceutically effective amountof a compound of formula (I)

or a pharmaceutically acceptable salt thereof, or a stereoisomer thereofor mixture of stereoisomers thereof, wherein R₁ is optionallysubstituted piperidinyl, optionally substituted pyridyl, optionallysubstituted pyrrolyl, optionally substituted pyrroldinyl,1,4-dimmethylthiazolyl, 2-ethyl-4-methylthiazolyl,2-isopropylthiazol-5-yl, thiazolyl, 3-ethylthiazol-5-yl,1-methylsulfonylpiperidin-4-yl, or R₁ is —C(O)Z, where Z is piperazinyl,(S)-2-(3-ethylpiperazin-1-yl), optionally substituted pyrrolopyrrolyl,piperidin-3-ylamino, or, R₁ is

 where R₁₃ is H, methylpyrazolyl, methylimidazolyl, benzyl,3-hydroxybutyl, 3-(dimethylamino)-2,2-dimethylpropyl, ethylamide,methylpyridyl, methylsulfonyl, (1-methylimidazol-2-yl)methyl,(1,5-dimethylimidazol-4-yl)methyl, (1-methylpyrrol-2-yl)methyl, or whereR₁₃ is C(O)W, where W is —N(CH₃)₂, piperidinyl, piperazinyl, ormorpholinyl, or, R₁ is

 where R₁₄ is C(O)CH₃, H, or (1-methylpyrrol-2-yl)methyl, or, R₁ is

 or R₁ is

 where A, B, and D may all be carbon, or where two of A, B, and D arecarbon and the other is nitrogen, or where one of A, B, and D is carbonand the remaining two are nitrogen; and when A is nitrogen R₄ is absent,when B is nitrogen R₂ is absent, and when D is nitrogen R₃ is absent;and wherein R₂ is H, —CH₃, or F, or, with R₃ and the atoms at positionsa and b, forms an optionally substituted pyridine or a pyrazole; andwherein R₃ is H, F, Cl, —CN, —CH₃, —OCH₃, —OH, —NH₂, methylsulfonyl,

or, with R₄ and the atoms at b and c, forms an optionally substitutedbenzene, optionally substituted imidazole, optionally substitutedpyrazole, optionally substituted pyrazolidine, optionally substitutedimidazolidine, optionally substituted isothiazole,

or, with R₂ and the atoms at a and b, forms an optionally substitutedpyridine or optionally substituted pyrazole; and wherein R₄ is F, —CN,—OCH₃, —OEt, H, Cl, Br, —NH—C(O)—CH—(CH₃)₂, —N(CH₃)₂, —CH₃, —CH₂OH,

optionally substituted piperazinyl, 4-hydroxypiperizin-1-yl, optionallysubstituted piperidinyl not attached to a phenyl group through anitrogen, or, with R₃ and the atoms at b and c, forms an optionallysubstituted pyrazole ring or

or, with R₅ and the atoms at c and d, forms an optionally substitutedpyrazole ring or an optionally substituted pyrrole ring, or, R₄ is-(q)-C(O)X, where q is a bond, is —NH—, or is —CH₂—, and where X is—NR₁₁R₁₂, where R₁₁ and R₁₂ are both H, both —CH₂CH₃, or both —CH₃, orwhere one of R₁₁ and R₁₂ is H and the other is 1,1-dimethylethyl,cyclobutyl, cyclopropyl, lower alkyl, methyl alcohol, propyl alcohol,cyclobutylmethyl; 2,3-dihydroxypropyl, benzyl, azetidinyl, pyrrolidinyl,piperidinyl, methylazetidinyl, —CH₂—NH—CH₃, pyrazolyl, piperazinyl,alcohol, —OCH₃, or

or where X is optionally substituted pyrrolidinyl attached through anitrogen to the carbonyl group of R₄, optionally substituted piperidinylnot attached through a nitrogen to the carbonyl group of R₄, optionallysubstituted pyrrolidinyl attached through a nitrogen to the carbonylgroup of R₄, optionally substituted piperazinyl attached through anitrogen to the carbonyl group of R₄, or optionally substitutedmorpholinyl attached through a nitrogen to the carbonyl group of R₄,

and wherein R₅ is H, F, Cl, —CH₃, —OCH₃, pyrrolyl,

or, with R₄ and the atoms at c and d, forms an optionally substitutedbenzene, an optionally substituted pyrazole, or an optionallysubstituted pyrrole, or, with R₆ and the atoms at d and e, forms anoptionally substituted pyridine, or R₅ is C(O)Y, where Y is —NH₂,—N(CH₃)₂, optionally substituted piperazinyl, optionally substitutedpiperidinyl,

and wherein R₆ is H, F, —CH₃, —CF₃, or, with R₅ and the atoms at c andd, forms an optionally substituted benzene or an optionally substitutedpyrazole; and wherein R₇ is —CF₃ or —CHF₂; and wherein R₈ is

and wherein R₉ is Br, Cl, F, I, or H; with the following provisos: whenR₄ is F: R₃ is not —CH₃ or F; R₃ is not —CH₃, —CN, F, Cl, or —OCH₃; R₅is not —CH₃, F, Cl, or —OCH₃; and R₆ is not —CH₃ or F; when R₄ is Cl: R₂is not F; R₃ is not F or —CN; R₅ is not F, —CN, or —C(O)N(CH₃)₂; R₆ isnot —CF₃ or F; D is not nitrogen; and either R₅ is —C(O)NH₂ or one ofR₂, R₃, R₅, and R₆ is —CH₃; when R₄ is —CH₃: R₃ is not F; R₅ is not F;and R₅ and R₆ do not form a pyrimidine together with the atoms at d ande; when R₄ is —OCH₃: R₃ is not F; R₃ is not Cl or —OCH₃, R₅ is not Cl or—OCH₃; and R₆ is not F or —CF₃; when R₄ is —CN: R₂ is not F; R₃ is notCl, F, or —OCH₃, R₅ is not Cl, F, or —OCH₃; and R₆ is not F; when R₄ is—OCH₂CH₃: R₃ is not Cl or F; R₅ is not Cl or F; and R₆ is not —CF₃; whenR₄ is

 R₃ is not H or F; and R₅ is not H or F; when R₄ is

 at least one of R₂, R₃, R₅ and R₆ is not H; when R₄ is

 R₃ is not F; and R₅ is not F; when R₂ is F: R₃ is not —OCH₃ or F; R₅ isnot —CN; and at least one of R₃, R₄, R₅, and R₆ is not H; when R₂ is Cl:R₃ is not F; when R₂ is —CH₃: R₃ is not Cl; at least one of R₃, R₄, R₅,and R₆ is not —CH₃; and R₄ and R₅ do not form a pyrazolyl with the atomsat c and d; when R₃ is —OCH₃: R₂ is not F; and R₆ is not F; when R₃ isF: R₂ is not —OCH₃; and X is not

when R₃ is Cl: R₅ is not Cl; R₁₁ is not benzyl; and R₁₂ is not benzyl;when R₅ is Cl, R₆ is not —CH₃; R₁₁ is not benzyl; and R₁₂ is not benzyl;when R₅ is F or —OCH₃: R₆ is not F; when R₆ is F: at least one of R₂,R₃, R₄, and R₅ is not H; when R₃ and R₅ are H: R₁₁ is not cyclopropyl;and R₁₂ is not cyclopropyl; when R₉ is Cl, R₁ is not an amide group;when B is nitrogen and A and D are carbon: R₄ may not be —CN or

when R₇ is —CHF₂ and R₄ is

 then R₄ does not have the absolute stereochemistry

 and wherein, when R₈ is

 then the following provisos are in effect; when R₄ is F: at least oneof R₂, R₃, R₅, and R₆ is not H; R₃ is not C(O)N(CH₃)₂; and R₅ is notC(O)N(CH₃)₂; when R₄ is Cl: at least one of R₂, R₃, R₅ and R₆ is not H;when R₃ is F: R₄ is not C(O)NHCH₂CH₂CH₂CH₃, C(O)N(CH₃)₂,C(O)NHCH₂CH₂CH₃, or C(O)NHC(CH₃)₃; R₄ is not C(O)NHCH₂CH₂CH₂OH,C(O)NHCH(CH₃)₂, —CN, or

R₁ is not

R₅ is not

R₃ is not

when R₂ is F: R₅ is not —C(O)NH₂; when R₂ is —CH₃, R₄ and R₅ do not forma pyrazole with atoms at c and d; and when B is nitrogen, R₃ and R₄ donot form an optionally substituted imidazole with the atoms at b and c;and wherein, when R₈ is

 then the following provisos are in effect; R₄ is not —CH₃,—C(O)NHCH₂CH₂OH, —NHC(O)CH(CH₃)₂, or

when R₄ is C(O)NHCH₃, at least one of R₂, R₃, R₅, and R₆ is not H; whenR₄ is —OCH₃: R₃ is not F or —CH₃; and R₅ is not F or —CH₃; when R₄ is

 R₃ is not Cl; and R₅ is not Cl; when R₄ is —C(O)NHCH(CH₃)₂ or—C(O)N(CH₂CH₃)₂; at least one of R₃ and R₅ is not H; R₅ is not —C(O)NH₂;and R₆ is not —CF₃.
 2. The method of claim 1, wherein said compound isadministered as a pharmaceutically acceptable salt.
 3. A method fortreatment of a systematic lupus erythematosus or lupus, comprisingadministering a pharmaceutically effective amount of4-((5S,7R)-5-(3,4-dimethoxyphenyl)-7-(trifluoromethyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidin-2-yl)phenyl)(piperazin-1-yl)methanoneor a pharmaceutically acceptable salt thereof.
 4. The method of claim 3,wherein said compound is administered as a pharmaceutically acceptablesalt.
 5. A method for treatment of a systematic lupus erythematosus orlupus, comprising administering a pharmaceutically effective amount of acompound of formula (IV)

or a pharmaceutically acceptable salt thereof, or a stereoisomer thereofor mixture of stereoisomers thereof, wherein: R_(7a) is H or F; andwherein Ring A is:

wherein Y₁ and Y₂ are independently selected from the group consistingof —CH₂— and —CH₂CH₂—, and wherein each of Y₁ and Y₂ is optionallysubstituted by C₁₋₃ alkyl;

wherein X₁, X₂, and X₃ are independently selected from the groupconsisting of —CH— and N;

wherein X₁, X₂, and X₃ are independently selected from the groupconsisting of —CH— and N;

wherein X is N or —CH— optionally substituted by —CH₃, F, or Cl, andwherein R₉ is —C(O)Z, wherein Z is 2,3-dihydroxypropylamine; a five toseven member cyclic diamine that is optionally bridged or optionallysubstituted at a carbon atom with a lower alkyl; a seven to ten memberbicyclodiamine; a seven to eleven member spirodiamine; —NH substitutedwith a four to seven member cyclic amine optionally substituted with—NH₂; —OH; —CH₂NHR, wherein R is H or lower alkyl; —NH substituted witha seven to eleven member spiroalkane optionally substituted with —NH₂;or R₉ is CH₃NHC(O)—, and a carbon atom on the aryl ring to which R₉ isattached is substituted with one of —CH₃, F, or Cl; R₉ is(CH₃)₂CHNHC(O)—, and a carbon atom on the aryl ring to which R₉ isattached is substituted with one of —CH₃, F, or Cl; or R₉ is(CH₃)₃CNHC(O)— and a carbon atom on the aryl ring to which R₉ isattached is substituted with one of —CH₃, F, or Cl; or R₉ is

 wherein the piperazine is optionally bridged or substituted with loweralkyl and R₁₀ is H or —CH₃; or R₉ is

 wherein n is 1-3 and the cyclic diamine is optionally bridged orsubstituted with lower alkyl; or R₉ is

 wherein n is 1-4; or R₉ is —NHC(O)NH₂, —CH₂C(O)NH— wherein the nitrogenis substituted with a four to seven member cyclic amine; —CH₂—C(O)—wherein the carbonyl is substituted with a seven to ten memberbicyclodiamine; and a four to seven member cyclic amine substituted with—CH₂C(O)NH₂; or

wherein X is N or —CH— wherein the C is optionally substituted by —CH₃,F, or Cl, and wherein R₁₀ is —C(O)NH— wherein the nitrogen issubstituted by a four to seven member cyclic amine; —C(O)— substitutedby a seven to ten member bicyclodiamine; —C(O)— substituted by a sevento eleven member spirodiamine; pyrazole; [1,2,4]oxadiazole optionallysubstituted by —CH₃ on a carbon atom of the oxadiazole; —NHC(O)CH₃;—CH₂— substituted by a piperazine; —CH₂— substituted by a piperazineincluding a methyl substituent; —C(O)— substituted by a five to sevenmember cyclic diamine; —C(O)NHCH₂— wherein the —CH₂— is substituted byazetidine; or —C(O)— substituted with a five to seven member cyclicamine wherein the amine includes an —NH₂ substituent; or cyanophenyl;isoquinoline; cyclohexene substituted with —NH₂ at the 4′ position;1,4-dimethylindazole-5-yl; 1,6-dimethylindazole-5-yl; cyclohexenesubstituted with spiropiperidine at the 4′ position;1-piperidinopyrazole; or o-methoxypyridine.
 6. The method of claim 5,wherein said compound is administered as a pharmaceutically acceptablesalt.